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The phenylpropanoid pathway is an important route of secondary metabolism involved in the synthesis of different phenolic compounds such as phenylpropenes, anthocyanins, stilbenoids, flavonoids, and monolignols. The flux toward monolignol biosynthesis through the phenylpropanoid pathway is controlled by specific genes from at least ten families. Lignin polymer is one of the major components of the plant cell wall and is mainly responsible for recalcitrance to saccharification in ethanol production from lignocellulosic biomass. Here, we identified and characterized sugarcane candidate genes from the general phenylpropanoid and monolignol-specific metabolism through a search of the sugarcane EST databases, phylogenetic analysis, a search for conserved amino acid residues important for enzymatic function, and analysis of expression patterns during culm development in two lignin-contrasting genotypes. Of these genes, 15 were cloned and, when available, their loci were identified using the recently released sugarcane genomes from Saccharum hybrid R570 and Saccharum spontaneum cultivars. Our analysis points out that ShPAL1, ShPAL2, ShC4H4, Sh4CL1, ShHCT1, ShC3H1, ShC3H2, ShCCoAOMT1, ShCOMT1, ShF5H1, ShCCR1, ShCAD2, and ShCAD7 are strong candidates to be bona fide lignin biosynthesis genes. Together, the results provide information about the candidate genes involved in monolignol biosynthesis in sugarcane and may provide useful information for further molecular genetic studies in sugarcane.

Dendrobium nobile is an orchid species highly popular for its therapeutic properties and is often used as a medicinal herb. Documenting miRNA-target associations in D. nobile is an important step to facilitate functional genomics studies in this species. Therefore, it is of interest to identify miRNA sequences from EST data available in public databases using known techniques and tools. We report 14 potential miRNAs from three ESTs of D. nobile. They belong to 3 miRNA families (miR390, miR528 and miR414) linking to transcription factor regulation, signal transduction, DNA and protein binding, and various cellular processes covering 34 different metabolic networks in KEGG. These results help in the understanding of miRNA-mRNAs functional networks in Dendrobium nobile.

Finger millet is a high nutritious cereal compared to maize, wheat, rice and sorghum and adaptable to different abiotic and biotic stresses. Understanding the molecular basis of unique traits of finger millets, is key in harnessing its potential as a nutritional security crop among other important aspects. In this study some accession from Africa and Asia were used to research the genetic diversity and population structure of finger millet using EST-SSR Markers. Twenty four accessions of finger millet were tested for polymorphism and highly polymorphic bands were generated in 27 EST markers. A total of 46 alleles were amplified and ranged from 2 to 3 with average of 1.703 per primer pair. The observed heterozygosity value of EST-SSR markers (mean = 0.004) was from 0 to 0.125 and the range of expected heterozygosity value was from 0.16 to 0.582 (mean=0.233). The range of PIC values were from 0.077 to 0.477 and the average PIC value was 0.273.The genetic relationship was divided into three major groups, with accessions from Africa showing a high level of polymorphism and unique population structure compared to Asian ones. These results echos the need for strategic continued colaborative breeding and other crop research programmes between Africa and Asia. The results from futher molecular evaluation will serve as important information for better and efficient management of genetic resources of finger millet for; conservation, crop improvement and intellectual property protection rights purposes.

Nuclear Expressed Sequence Tag (EST) microsatellite markers were developed for the Tasmanian palaeoendemic conifer Lagarostrobos franklinii (Hook.-f.) Quinn for genetic studies. RNAseq data was mined for EST microsatellites, and primer pairs were synthesised from 70 contigs with 50 producing amplification products. Of these 50, 10 reliably amplified and displayed polymorphism across 8 samples representing the entire species range. The genetic diversity of these 10 loci was then examined in three wild populations (84 samples). The number of alleles varied from two to thirteen per locus with the average number of alleles per population ranging between 3.0 – 4.7. Observed and expected heterozygosity ranged from 0.34 – 0.42 and 0.37 – 0.44, respectively. Marker cross-amplification was tested in the New Zealand sister species Manoao colensoi (Hook. f.) Molloy, but no markers amplified reliably, which possibly reflects the age of divergence between these species (~64 million years). These are the first microsatellite markers developed for the monotypic genus Lagarostrobos. They will be valuable for assessing the species extant genetic diversity, the impact of past climatic perturbations and human disturbance and the role of clonal propagation in recruitment.

Olive is considered one of the oldest and the most important cultivated fruit trees in Albania. In the present study, the genetic diversity and structure of Albanian olive germplasm is represented by a set of 194 olive genotypes collected in-situ in their natural ecosystems and in the ex-situ collection. The study was conducted using 26 microsatellite markers (14 genomic SSR and 12 Expressed Sequence Tag microsatellites). The identity analysis revealed 183 unique genotypes. Genetic distance-based and model-based Bayesian analyses were used to investigate the genetic diversity, relatedness, and the partitioning of the genetic variability among the Albanian olive germplasm. The genetic distance-based analysis grouped olives into 12 clusters, with an average similarity of 50.9%. Albanian native olives clustered in one main group separated from introduced foreign cultivars, which was also supported by Principal Coordinate Analysis (PCoA) and model-based methods. A core collection of 57 genotypes representing all allelic richness found in Albanian germplasm was developed for the first time. Herein, we report the first extended genetic characterization and structure of olive germplasm in Albania. The findings suggest that Albanian olive germplasm is a unique gene pool and provides an interesting genetic basis for breeding programs.

Wild barley, Hordeum marinum subsp. gussoneanum (2n = 28) is a valuable source of genes that determine resistance to abiotic stresses. These resistance traits might be transferred to wheat due to the crossability of wild barley with bread wheat. The availability of reliable and rapid methods for the identification of H. marinum subsp. gussoneanum chromatin in a wheat background would facilitate the development of introgression wheat genotypes. For this purpose, we evaluated the applicability of eighty-seven H. vulgare EST markers for studying bread wheat – H. marinum subsp. gussoneanum substitution and addition lines. Of all of the markers studied, forty-three (49%) were amplified in H. marinum ssp. gussoneanum and wheat introgression lines. The identification of wild barley chromosomes using EST markers confirmed the GISH and C-banding data. Thus, it was established that the H. vulgare EST markers can be successfully used to identify the chromosomes of the H. marinum subsp. gussoneanum in introgression lines of wheat.

BackgroundOverlapping genes share same genomic regions in parallel (sense) or anti-parallel (anti-sense) orientations. These gene pairs seem to occur in all domains of life and are best known from viruses. However, the advantage and biological significance of overlapping genes is still unclear. Expressed sequence tags (ESTs) analysis enabled us to uncover an overlapping gene pair in the human genome.ResultsBy using in silico analysis of previous experimental documentations, we reveal a new form of overlapping genes in the human genome, in which two genes found on opposite strands (Pou5f1andTcf19), share two exons and one intron enclosed, at the same positions, between OCT4B3 and TCF19-D splice variants.ConclusionsThis new form of overlapping gene expands our previous perception of splicing events and may shed more light on the complexity of gene regulation in higher organisms. Additional such genes might be detected by ESTs analysis also of other organisms.

Compared to cereal crops several legumes are less characterized at the genomic level and rightly referred as orphan crops. Transfer of knowledge between model and crop legumes allows development of orthologous pan-taxon genomic tools to benefit research on resource poor taxa. Here, we developed 278 intron flanking gene-specific markers by BLAST aligning pigeonpea (Cajanus cajan (L.) Millsp.) expressed sequence tags (ESTs) with complete genome sequence of barrel medic (Medicago truncatula). A random 192 PCR primer pairs representing loci across the haploid genome (n = 8) of barrel medic were tested on a few important members of legume family. The single copy amplification rates of 31.8% (peanut, Arachis hypogaea) to 77.6% (barrel medic) signifies the success of cross taxon primers and suggested their potential use in comparative legume genomics. Genetic diversity was assessed in 48 pigeonpea genotypes using 143 intron flanking markers which revealed 71 polymorphic markers with PIC values ranging from 0.04 to 0.45 with an average of 0.23 per marker. The PCR products of different varieties of pigeonpea, cowpea and chickpea were sequenced and aligned to find putative SNPs. Integration of these newly developed markers into genetic maps in resource poor legumes will not only aid in the map saturation but also in designing successful marker-assisted selection programmes.

Gentiana is an important genus of around 360 medicinally important species, majority of which are not well characterized. Despite its importance, very few genomic resources are available for Gentiana L. Till date, the number of informative and robust simple sequence repeat (SSR)-based markers is limited and very few efforts have been made for their development. A set of robust, freely accessible and informative SSR markers for Gentiana is a pre-requisite for any molecular systematic as well as improvement studies in this group of pharmacologically valuable plants. In view of the importance of these plants, Expressed Sequence Tag (EST) sequences of 18 Gentiana species were surveyed for the development of a large set of non-redundant SSR markers. A total of 5808 perfect SSR with an average length of 17 bp and relative abundance of 214 loci/Mb were identified in the analysed 47,487 EST sequences using Krait software. Mapping of the ESTs resulted in gene ontology annotations of 49.14% of the sequences. Based on these perfect SSRs, 2902 primer pairs were designed, and 60 markers were randomly selected and validated on a set of Gentiana kurroo Royle accessions. Among the screened markers, 39 (65%) were found to be cross-species transferable. This is the first report of the largest set of functional, novel genic SSR markers in Gentiana, which will be a valuable resource for future characterization, genotype identification, conservation and genomic studies in the various species of this group of important medicinal plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02969-4.

The domesticated silkworm, Bombyx mori, is an important model system for the order Lepidoptera. Currently, based on third-generation sequencing, the chromosome-level genome of Bombyx mori has been released. However, its transcripts were mainly assembled by using short reads of second-generation sequencing and expressed sequence tags which cannot explain the transcript profile accurately. Here, we used PacBio Iso-Seq technology to investigate the transcripts from 45 developmental stages of Bombyx mori. We obtained 25,970 non-redundant high-quality consensus isoforms capturing ∼60% of previous reported RNAs, 15,431 (∼47%) novel transcripts, and identified 7,253 long non-coding RNA (lncRNA) with a large proportion of novel lncRNA (∼56%). In addition, we found that transposable elements (TEs) exonization account for 11,671 (∼45%) transcripts including 5,980 protein-coding transcripts (∼32%) and 5,691 lncRNAs (∼79%). Overall, our results expand the silkworm transcripts and have general implications to understand the interaction between TEs and their host genes. These transcripts resource will promote functional studies of genes and lncRNAs as well as TEs in the silkworm.

Ginger (Zingiber officinale Rosc.) is an economically important and valuable spice crop around the world. It is used as food, spice, condiment, and medicine. A considerable extent of genetic diversity in ginger occurs in the Western Ghats and North-Eastern India. However, genetic diversity studies at the molecular level in ginger is limited due to limited availability of genetic and genomic information. In the present study, for the first time, we have identified and validated expressed sequence tag (EST)-simple sequence repeat (SSR) markers from ginger. We obtained 16,790 EST-SSR loci from 78987 unigenes, and 4597 SSR loci in the predicted 76929 coding sequences from RNA-Seq assembled contigs of ginger through Illumina paired-end sequencing. Gene ontology results indicate that the unigenes with SSR loci participate in various biological processes such as metabolism, growth, and development in ginger. One hundred and twenty-five primer pairs were designed from unigenes and coding sequences. These primers were tested for PCR optimization, characterization, and amplification and identified 12 novel EST-SSR markers. Twelve flanking polymorphic EST-SSR primers were validated using 48 ginger genotypes representing North-Eastern India and different eco-geographical adaptations by PCR amplification and allele sizing through capillary electrophoresis. Twelve EST-SSR primers generated a total of 111 alleles with an average of 9.25 alleles per locus and allele sizes ranging between 115-189bp. This study implies that the SSR markers designed from transcriptome sequences provides ample EST-SSR resources, which are helpful for genetic diversity analysis of Zingiberaceae species and molecular verification of ginger genotypes.

In the course of our search for the gene responsible for X-linked cone-rod dystrophy (COD1), we constructed a physical map and contig (encompassing the region between DXS556 and DXS228), and identified sequences showing homologies to the expressed sequence tags (ESTs) that matched CRSP2 (EXLM1) transcript. We confirmed the expression of the CRSP2 gene in the retina and refined its exact genomic location between DXS1368 and DXS993. We demonstrated that the entire transcript is encoded within 31 exons. Primers were designed for mutation analysis of the exons by direct sequencing of PCR products from genomic DNA, and revealed no mutations in COD1 families. We subsequently excluded CRSP2 as a candidate for COD1 by demonstrating the causative mutations in the RPGR. However, due to its expression in different tissues and its contribution to transcriptional regulation, CRSP2 may be a candidate for other diseases that map to this region of the X chromosome.

Summary Drug discovery targeting G protein-coupled receptors (GPCRs), the largest known class of therapeutic targets, is challenging. To facilitate the rapid discovery and development of GPCR drugs, we built a system, PanGPCR, to predict multiple potential GPCR targets and their expression locations in the tissues, side effects and possible repurposing of GPCR drugs. With PanGPCR, the compound of interest is docked to a library of 36 experimentally determined crystal structures comprising of 46 docking sites for human GPCRs, and a ranked list is generated from the docking studies to assess all GPCRs and their binding affinities. Users can determine a given compound’s GPCR targets and its repurposing potential accordingly. Moreover, potential side effects collected from the SIDER (Side-Effect Resource) database and mapped to 45 tissues and organs are provided by linking predicted off-targets and their expressed sequence tag profiles. With PanGPCR, multiple targets, repurposing potential and side effects can be determined by simply uploading a small ligand. Availability and implementation PanGPCR is freely accessible at https://gpcrpanel.cmdm.tw/index.html. Supplementary information Supplementary data are available at Bioinformatics online.

Salinization of soil is a prime abiotic stress that limits agriculture productivity worldwide. To Study the mechanisms that halophytes take up to survive under high salt condition is important in engineering salinity stress tolerance in sensitive species. Suaeda nudiflora is a halophyte plant that grows in the saline environment and extreme high tidal belt. The species have high capability to produce high protein biomass in salty soils due to C4 photosynthesis. The physiological and biochemical changes in S. nudiflora under salinity stress were studied by measuring chlorophyll content, electrolytic leakage, level of lipid peroxidation and total soluble sugars. Increased lipid peroxidation and electrolytic leakage was observed in salt stressed S. nudiflora compared to control plants. A suppression subtractive hybridization strategy was employed to identify differentially expressed genes under salt treatment in S. nudiflora. A total of 333 positive clones were identified and screened. Of these, 250 expressed sequence tags were identified. cDNA subtraction library resulted in 33 contigs and 138 singletons. The functional annotation and metabolic pathways identification were performed using the Blast2GO program. In addition, we analyzed the expression patterns of 18 genes associated with salt stress-responsive pathways by semi-quantitative PCR under salt and elevated carbon dioxide (CO2) conditions. Several of the analyzed genes showed an increase in expression levels under different time points of salt treatment and at different concentrations of salt. When the same genes were studied for its expression under elevated CO2 concentrations, most of the known salt responsive genes showed higher expression under the combined treatment of elevated CO2 concentrations (500 ppm) and NaCl treatment (200 mM) compare to ambient condition. This implies that salt responsive genes are enhanced at elevated CO2 concentrations.

The abundance and the diversity of oligo- and polysaccharides provide a wide range of biological roles attributed either to these carbohydrates or to their relevant enzymes, i.e., the glycoside hydrolases (GHs). The biocatalysis by these families of enzymes is highly attractive for the generation of products used in potential applications, e.g., pharmaceuticals and food industries. It is thus very important to extract and characterize such enzymes, particularly from plant tissues. In this study, we characterized novel sequences of class I chitinases from seedlings extract of the common oat (Avena sativa L.) using proteomics and sequence-structure-function analysis. These enzymes, which belong to the GH19 family of protein, were extracted from oat and identified using SDS-PAGE, trypsin digestion, LC-MS-MS, and sequence-structure-function analysis. The amino acid sequences of the oat tryptic peptides were used to identify cDNAs from the Avena sativa databases of the expressed sequence tags (ESTs) and transcriptome shotgun assembly (TSA). Based upon the Avena sativa sequences of ESTs and TSA, at least 4 predicted genes that encoded oat class I chitinases were identified and reported. The structural characterization of the oat sequences of chitinases provided valuable insights to the context.

Objective: Brain tumor-initiating cells are characterized by their features of self-renewal, multi-lineage differentiation, and tumorigenicity. We analyzed the gene expression of brain tumor-initiating cells to identify their novel cellular markers. Methods: We performed cDNA microarray, in silico expressed sequence tags (ESTs), RT-PCR, and q-PCR analyses. Results: We identified 10 genes that were more highly expressed in brain tumor-initiating cells than in neural stem cells. In addition, we identified 10 other genes that were more highly expressed in brain tumor-initiating cells than in glioma cell line cells from the cDNA microarray analysis. Using the EST database, we looked to see if the 20 genes were expressed more highly in gliomas, compared with normal adult brains. Among the 20 genes, five (KLRC2, HOXB2, KCNJ2, KLRC1, and COL20A1) were expressed more than twice in glioma samples, compared with normal adult brains, and, therefore, were referred for further evaluation. RT-PCR was conducted using cDNA samples obtained from neural stem cells, normal brain tissue, fetal brain tissue, glioma cell lines, and glioma tumor-initiating cell lines. KLRC2, a transmembrane activating receptor in natural killer cells, was expressed more highly in glioma-initiating cells than in neural stem cell lines or normal adult brain tissue. The q-PCR analysis revealed that expression of KLRC2 was significantly higher in brain tumor-initiating cells compared to normal brain controls. Conclusion: KLRC2 could be a novel cellular marker for brain tumor-initiating cells.

Gobiobotia filifer is a small benthic fish distributed in Yangtze River Basin. The abundance of G. filifer increased after impoundment of Xiluodu Dam and Xiangjiaba Dam. The state of population structure and changes of genetic diversity before and after impoundment of Xiluodu Dam and Xiangjiaba Dam were interesting issues. However, efficient molecular markers were rare, which will limit us to solve above problems. Twenty‐eight expressed sequence tag SSRs (EST‐SSRs) were successfully identified and verified as stable amplification and polymorphic loci by polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The number of alleles at these EST‐SSR loci ranged from 3 to 14, the polymorphism information content values were 0.125–0.897, and the observed and expected heterozygosities were 0.0–0.857 and 0.132–0.928, respectively. Cross‐species amplification of the 28 loci developed in this study was examined in seven individuals of each of the 7 taxa. The amplification efficiency of 28 EST‐SSRs primer pairs is related to the distance of genetic relationship between cross‐species with G. filifer, and same subfamily species (Xenophysogobio boulengeri and Xenophysogobio nudicorpa) showed the highest (50%) amplification efficiency. These EST‐SSR markers could be used to analyse genetic diversity and population structure of G. filifer and related species.

The genus Hibiscus comprises approximately 300 diverse species whose genetic relationships have not been extensively clarified. In this study, we evaluated the cross-species transferability of 102 expressed sequence tag-simple sequence repeat (EST-SSR) markers derived from the transcriptome of kenaf H. cannabinus L. and used them to assess the genetic diversity and inter-relationships of 87 accessions and 7 cultivars of 18 Hibiscus species. Among 102 markers, 100 (98%) were polymorphic in these 94 accessions/cultivars. The rates of cross-species transferability of the EST-SSR markers ranged from 83 H. trionum L. to 99% H. ponticus Rupr., with an average of 89.9%. A total of 871 alleles were detected using the 100 polymorphic EST-SSR markers; the number of alleles ranged from 2 (RBRC_Hc_ES_76) to 16 (RBRC_Hc_ES_80) and averaged 8.71. Polymorphism information content values varied from 0.15 (RBRC_Hc_ES_74) to 0.84 (RBRC_Hc_ES_3, RBRC_Hc_ES_95), with an average of 0.56. Phylogenetic and population structure analyses classified the 94 accessions/cultivars into two and two or three groups, respectively. Overall, our results demonstrate that the EST-SSR markers derived from the kenaf transcriptome were applied successfully to other Hibiscus species to provide useful information on the genetic diversity of this genus.

Onion (Allium cepa L.) is a popular spice and a plant of high medicinal value. Conventional breeding and genetic improvement efforts were largely limited due to self-incompatibility and heterozygosity. Recently, marker assisted breeding has significantly reduced time and labour in developing elite varieties. But very limited polymorphic and cross-transferable markers are available in onion. There is an urgent need to develop polymorphic markers in Allium to expedite and introgress desirable traits from wild relatives (which are rich bioresource of various biotic and abiotic resistance genes) to A. cepa. Considering limited availability of reliable molecular markers in Allium and wild relatives, in current study, 20,204 ESTs (3750 contigs and 8364 singletons), of A. cepa were successfully utilized for identification of over 2689 intron length polymorphic (ILP) markers. A set of 30 markers was tested for polymorphism in onion and cross-transferability in garlic and related wild species. Among these, eighteen markers amplified at least one of the accessions of A. cepa. Transferability of these ILP markers was ranged from 21.7 to 95.7% in Allium spp. Low level of polymorphism in A. cepa compared to wild Allium species is reported. Based on the Jaccard dissimilarity matrix, a neighbour-joining tree was constructed, which clustered all the 23 varieties/accessions under three groups. All the varieties of A. cepa were clearly clustered separately under group I. However, there was intermixing of varieties/accessions of A. sativum L. and wild relatives, which may possibly be due to less number of markers validated for cross-transferability. In future, larger set of markers will be used to resolve the genetic variations among wild varieties and A. sativum These 18 polymorphic ILP markers could be utilised for diversity characterization of Allium spp., varietal identification, mapping of genes and introgression of desirable traits from wild relatives.

Background and objectives: Liver and esophageal cancers are common among the Iranian population. This study aims to explore the common up-regulated genes in liver and esophageal cancer tissues using expressed sequence tags (ESTs) and to identify the role of key genes in cancer development. Methods: EST profiles of protein-coding genes in normal and cancerous hepatic and esophageal tissues were extracted from the UniGene database. Genes with > 1500 transcripts per million were selected as highly expressed. The cancer to normal ratio of up-regulated genes was calculated. The shared overexpressed genes between liver and esophageal cancer tissues were determined. Finally, functional classification and pathway analysis were performed on the genes using the STRING and Enrichr databases. Results: Of 17,242 genes, 53 and 26 genes were overexpressed in the liver and esophageal cancer tissues, respectively. Nine up-regulated genes (APLP2, EEF1G, ENO1, HSP90AA1, HSP90AB1, HSPA8, KRT18, RPL4 and UBC) were shared between the two cancer tissues, which were involved in cell cycle progression through G2/M checkpoint, G1/S transition and DNA replication. They were also involved in the vascular endothelial growth factor, hypoxia-inducible factor 1 and estrogen signal pathways as well as the Toll-like receptor cascade. Conclusion: Based on the results, the identically up-regulated genes and underlying molecular mechanisms implicated in both cancers could be valuable targets for diagnosis and treatment of cancer.

Heterostylous Primula forbesii is an important ornamental flower in China because of its long-lasting flowers and winter bloom. This study aimed to develop markers of expressed sequence tag–simple sequence repeats (EST-SSRs) that are associated with heterostyly and that can be used for molecular-assisted selective breeding in P. forbesii. We investigated 114,474 unigenes and identified 25,095 SSRs in P. forbesii. Dinucleotide repeats (46.14%), mononucleotide repeats (44.65%), and trinucleotide repeats (8.27%) were the most abundant SSRs. Among the 25,095 SSRs, 10,645 SSR primer pairs were successfully designed, of which 130 primer pairs were randomly selected for further amplification validation using eight accessions of P. forbesii; 98 pairs produced clear and stable polymerase chain reaction (PCR) products, and 28 pairs showed polymorphism. Bulked segregant analysis (BSA) was conducted for the F1 population with respect to thrum style and pin style by scanning 28 polymorphic SSR primer combinations. One SSR marker, c64326, linked to the heterostyly trait at a genetic distance of ≈3.70 cM was identified. The marker c64326 was further validated in two populations with an accuracy of 97.92% and 90.63%. The novel and linked EST-SSR markers can be valuable resources for genetic diversity analysis, mapping, and marker-assisted breeding in P. forbesii.

Disease outbreaks devastate Pyropia aquaculture farms every year. The three most common and serious diseases are Olpidiopsis-blight and red-rot disease caused by oomycete pathogens and green-spot disease caused by the PyroV1 virus. We hypothesized that a basic genetic profile of molecular defenses will be revealed by comparing and analyzing the genetic response of Pyropia tenera against the above three pathogens. RNAs isolated from infected thalli were hybridized onto an oligochip containing 15,115 primers designed from P. tenera expressed sequence tags (EST)s. Microarray profiles of the three diseases were compared and interpreted together with histochemical observation. Massive amounts of reactive oxygen species accumulated in P. tenera cells exposed to oomycete pathogens. Heat shock genes and serine proteases were the most highly up-regulated genes in all infection experiments. Genes involved in RNA metabolism, ribosomal proteins and antioxidant metabolism were also highly up-regulated. Genetic profiles of P. tenera in response to pathogens were most similar between the two biotrophic pathogens, Olpidiopsis pyropiae and PyroV1 virus. A group of plant resistance genes were specifically regulated against each pathogen. Our results suggested that disease response in P. tenera consists of a general constitutive defense and a genetic toolkit against specific pathogens.

MicroRNAs (miRNAs) are conserved small non coding RNAs, which are typically 22-24 nucleotides long and play an important role in post transcription regulation andin various biological processes in both animals and plants. Ocimum basilicum is an important medicinal plant having different bioactive compounds eugenol and essential oils that possess numerous therapeutic properties. However, only a few miRNAs of Ocimum basilicum and its function have been studied till date. The present study focusses on the identification of miRNA from expressed sequenced tags by carrying out computational approaches based on the homology search method. A total of 10 potential miRNAs with 8 different families were predicted in O.basilicum. Furthermore, the psRNA target server was used to predict cross kingdom target genes on human transcriptome for identification ofpotential miRNAs. Eight miRNA families were found to modulate the 87 human target genes which were associated with RAS/MAPK signalling cascade, cardiomyopathy, HIV, breast cancer, lung cancer, Alzheimer's diseases and several neurological disorders. Moreover, O.basilicum miRNAs regulate the key human target genes having significance in various diseases and important biological networks with 10 hub nodes interactions. Thus this study gives the pave for further studies to explore the potential of miRNA mediated cross kingdom regulation and treatment of various diseases including cancer.

Key message An EST-SSR database of Magnolia wufengensis was first generated by transcriptome sequencing, the SSRs related to flower pigment synthesis were validated, and the transferability of the EST-SSR markers to other species was additionally investigated. Magnolia (M.) wufengensis is a distinct ornamental species endemic to China and is famous for its gorgeous flower color. However, the limitation of understanding genetic resources has restricted its conservation and breeding. This study used RNA-Seq to generate thousands of expressed sequence tags (ESTs) with which thousands of functional markers were developed. Of the 9567 simple sequence repeats (SSRs) identified, 8306 contained unigene sequences. Among the different SSR motif types, dinucleotide repeats (50.8%) were the most abundant, followed by trinucleotide repeats (32.4%). AG/CT (37.8%) and AAG/CTT (11.8%) were the most enriched repeat motifs. A total of 5203 EST-SSR novel primer pairs were designed to construct a reference database, from which 33 related to floral pigment formation were PCR-validated by testing them on the five cultivars of M. wufengensis. Twenty-two primer pairs displayed clear PCR products in the verification. Moreover, the number of alleles per marker ranged from 1 to 6 with an average of 3.41, and the polymorphic bands ranged from 0 to 6 with an average of 2.77. Additionally, the genetic distances were positively correlated with certain phenotypic indexes, including anthocyanin and flavonoid contents. Furthermore, applying these EST-SSR markers to other related species showed that 50.0–68.1% of the markers were transferable. The identified EST-SSRs will not only inspire future molecular-assisted breeding in Magnoliaceae but also facilitate targeted research in marker-trait association for floral pigment formation and genetic diversity analysis.

Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.

Background: A total of 62,591 cowpea expressed sequence tags (ESTs) were BLAST aligned to the whole-genome sequence of barrel medic (Medicago truncatula) to develop conserved intron scanning primers (CISPs). The efficacy of the primers was tested across 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea. Genetic diversity was assessed using the same primers on different cowpea genotypes. Single-nucleotide polymorphisms (SNPs) were detected, which were later converted to length polymorphism markers for easy genotyping. CISPs developed in this study were used in tagging resistance to bacterial leaf blight disease in cowpea. Results: A total of 1262 CISPs were designed. The single-copy amplification success rates using these primers on 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea were approximately 60% in most of the legumes except soybean (47%) and peanut (37%). Genetic diversity analysis of 35 cowpea genotypes using 179 CISPs revealed 123 polymorphic markers with PIC values ranging from 0.05 to 0.59. Potential SNPs identified in cowpea, chickpea, and pigeon pea were converted to PCR primers of various sizes for easy genotyping. Using the markers developed in this study, a genetic linkage map was constructed with 11 linkage groups in cowpea. QTL mapping with 194 F3 progeny families derived from the cross C-152 × V-16 resulted in the identification of three QTLs for resistance to bacterial leaf blight disease. Conclusions: CISPs were proved to be efficient markers to identify various other marker classes like SNPs through comparative genomic studies in lesser studied crops and to aid in systematic sampling of the entire genome for well-distributed markers at low cost.How to cite: Bi R, Chandappa LH, Siddalingaiah L, et al. Leveraging barrel medic genome sequence for the development and use of genomic resources for genetic analysis and breeding in legumes. Electron J Biotechnol 2019;39. https://doi.org/10.1016/j.ejbt.2019.02.002 Keywords: Barrel medic, Cowpea, Comparative genomics, ESTs, BLAST, CISPs, SNPs, Linkage map, QTL, Bacterial blight, Legumes

Six mandarin cultivars, Ponkan (Citrus reticulata), Willowleaf (Citrus deliciosa), Kinnow (Citrus nobilis × C. deliciosa), Murcott (purported C. reticulata × Citrus sinensis), W. Murcott [purported (C. reticulata × C. sinensis) × C. reticulata)], and Snack (purported C. reticulata hybrid), were used in protoplast fusion with different parental combinations to generate somatic hybrids. Sixty-five somatic regenerants were obtained using optimized formulation of enzymes and molecular weight of polyethylene glycol for improved protoplast yield and heterokaryon fusion rate, respectively. Flow cytometry was used to determine the ploidy level of somatic regenerants, and nuclear expressed sequence tag–simple sequence repeat (EST-SSR) markers to determine their parental source. Of the 65 somatic regenerants, 46 were identified as autotetraploids, 18 allotetraploids, and one undefined. The EST-SSR markers also revealed that some ‘W. Murcott’ embryogenic callus lines that were presumed to be of nucellar origin were actually derived unexpectedly from individual ovules of zygotic origin. These mandarin-derived tetraploids are valuable as potential breeding parents for interploid crosses with an aim at seedlessness and easy-peeling traits.

Betula utilis D. Don. is an important species of alpine Himalaya and forms the major treeline component of western Himalaya. The different populations of B. utilis are declining and are under high risk. In the present study, novel expressed sequence tag–simple sequence repeat (EST–SSR) primers were developed from expressed sequence tag (EST) data of different Betula species. Of the 10,796 designed primers, the percentages of di-, tri-, tetra-, penta-, and hexa-repeats were 36%, 35%, 15%, 5.5% and 7.7%, respectively. For validation, 50 primers were synthesized randomly and were characterized in 20 different B. utilis accessions from north-western Himalaya. Of these, 45 primers amplified fragments in a range of 1–6. The 24 polymorphic primers produced 111 fragments in aggregate with 4.6 fragments on average. Polymorphism information content (PIC) ranged from 0.288 in marker BUMS-24 to 0.497 in BUMS-3 and BUMS-7, with an average of 0.447 among polymorphic markers. Dendrogram based on Jaccard’s similarity coefficient and UPGMA method showed that newly developed SSR markers distinguished twenty accessions of B. utilis into two groups. As no SSR markers were available in this species, the newly developed markers will foster molecular genetics research and conservation efforts for this species.

Background Controlled turnover of proteins as mediated by the ubiquitin proteasome system (UPS) is an important element in plant defense against environmental and pathogen stresses. E3 ligases play a central role in subjecting proteins to hydrolysis by the UPS. Recently, it has been demonstrated that a specific class of E3 ligases termed the U-box ligases are directly associated with the defense mechanisms against abiotic and biotic stresses in several plants. However, no studies on U-box E3 ligases have been performed in one of the important staple crops, barley. Results In this study, we identified 67 putative U-box E3 ligases from the barley genome and expressed sequence tags (ESTs). Similar to Arabidopsis and rice U-box E3 ligases, most of barley U-box E3 ligases possess evolutionary well-conserved domain organizations. Based on the domain compositions and arrangements, the barley U-box proteins were classified into eight different classes. Along with this new classification, we refined the previously reported classifications of U-box E3 ligase genes in Arabidopsis and rice. Furthermore, we investigated the expression profile of 67 U-box E3 ligase genes in response to drought stress and pathogen infection. We observed that many U-box E3 ligase genes were specifically up-and-down regulated by drought stress or by fungal infection, implying their possible roles of some U-box E3 ligase genes in the stress responses. Conclusion This study reports the classification of U-box E3 ligases in barley and their expression profiles against drought stress and pathogen infection. Therefore, the classification and expression profiling of barley U-box genes can be used as a platform to functionally define the stress-related E3 ligases in barley. Electronic supplementary material The online version of this article (10.1186/s12864-019-5696-z) contains supplementary material, which is available to authorized users.

Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify β-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as β-amylases. Insights into structural characterization of the oat predicted β-amylases were analyzed using in silico approaches. The identified β-amylases conserved the two Glu residues assigned as the “putative” catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (β/α)8-barrel architecture was found in the predicted oat β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)8-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat β-amylases and thus confirming that are exo-acting hydrolases. The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.

Background:MicroRNAs (miRNAs) are a class of small non-coding, endogenous RNAs that regulate gene expression at post-transcriptional level. In plants, miRNAs are usually of 18-24 nucleotide in length and play humongous role by aiding in development, growth, defense, biotic and abiotic stress responses, etc.Objective:Arachis hypogaea is an economically important oil seed crop and human dietary source cultivated mostly in tropical and subtropical regions. In the present study, an initiative was taken to uncover miRNAs, their targets and functions in this important plant species. Method: Comparative genomics strategy coupled with bioinformatics approaches was deployed for the identification of miRNAs, their corresponding targets and functions by exploiting biological databases and tools.Results:The study was able to identify 34 conserved miRNA candidates, belonging to 17 miRNA families, contributed by 23 and 3 precursor miRNAs from A. hypogaea Expressed Sequence Tags (EST) and Genome Survey Sequences (GSS), respectively. As well, 495 EST and 917 unigene sequences were predicted as targets for the identified miRNAs. Herein, psRNAtarget server and TargetFinder tool were used to predict unigene targets, whereas comparative genomics strategy was used for identifying EST targets. Functional annotation of the identified targets revealed that the identified miRNAs regulate mRNAs that participate in key biological and metabolic processes. Pathway enrichment analysis using KEGG database also revealed that they regulate important metabolic pathways including antibiotic biosynthesis, biosynthesis of unsaturated fatty acids, amino acids metabolism and flavonoid biosynthesis.Conclusion:The outcome of the study would aid experimental biologists to focus on these miRNAs to facilitate improved crop development and yield.

Genetic mapping for faba bean lags far behind other major crops. Density enhancement of the faba bean genetic linkage map was carried out by screening 5,325 genomic SSR primers and 2033 expressed sequence tag (EST)‐SSR primers on the parental cultivars '91825' and 'K1563'. Two hundred and fifteen genomic SSR and 133 EST‐SSR primer pairs that detected polymorphisms in the parents were used to screen 129 F₂ individuals. This study added 337 more SSR markers and extended the previous linkage map by 2928.45 cM to a total of 4516.75 cM. The number of SSR markers in the linkage groups varied from 12 to 136 while the length of each linkage group ranged from 129.35 to 1180.21 cM. The average distance between adjacent loci in the enhanced genetic linkage map was 9.71 cM, which is 2.79 cM shorter than the first linkage map of faba bean. The density‐enhanced genetic map of faba bean will be useful for marker‐assisted selection and breeding in this important legume crop.

Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a full‑length enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5'End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 high‑quality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (P‑value

Sugarcane expressed sequence tags (ESTs) database (db) offers a great opportunity for developing functional molecular markers associated with the desirable agronomic traits. The aim of the present study was to develop informative simple sequence repeat (SSR) markers in sugarcane using sugarcane expressed sequence tags (ESTs), characterization with respect to their abundance, distribution, variability, and to test their competence in genetic diversity. A total of 361 EST-SSR markers were developed and screened for their ability to detect polymorphism across the wide range of sugarcane germplasm. Among the developed SSRs, TNRs containing motifs were predominant with a frequency of 55%, which followed by TtNRs (22%), PNRs (11%), DNRs (7%), and HNRs (5%). Gene Ontology and homology search was conducted to establish the relevancy of SSRs in functional genomics. Resulting, ∼39% SSRs were found to be functional and confirmed homology with different proteins and transcription factors involved in gene expression. A set of 96 polymorphic primer pairs was used to evaluate genetic diversity among Saccharum species, related genera and cross amplification in other cereal grasses. A total of 710 alleles were generated which ranged from 2 to 15 with an average of 7.40 alleles per locus and showed 100% polymorphism. Polymorphism information content (PIC) value was ranged from 0.35 to 0.91 with an average of 0.71. Majority of the markers developed in current study were related to sucrose metabolism, stress physiology, plant immune responses, signal transduction, biosynthesis of secondary metabolites, ATP generation, growth and other biochemical process. The high degree of polymorphism, cross-species amplification and genetic diversity assessed with these SSR markers suggested their utility for genome mapping, QTL analysis, comparative genomics, gene-based association studies, and marker-assisted selection in sugarcane. Moreover, these informative EST-SSRs described here would enrich existing repertoire of genetic resources for sugarcane and also other related taxa constitute the important secondary genepool for sugarcane genetic improvement.

We developed 10 novel simple sequence repeat markers from expressed sequence tags for the honey locust seed beetle, Megabruchidius dorsalis Fahraeus 1839 (Coleoptera: Bruchidae, using de novo transcriptome analysis based on next-generation sequencing. In a M. dorsalis Harataima (Kanagawa-pref.) population, the number of alleles per locus ranged from 2 to 6, with an average of 4.0. Observed and expected heterozygosities ranged from 0.13 to 0.72 and 0.17 to 0.72, respectively. We initially developed 11 novel markers, but one was eliminated because it showed significant linkage disequilibrium with another locus after Bonferroni correction. To check the applicability of the remaining 10 markers, we used them to analyze two additional geographic populations (Yashima, Akita-pref., and Kameoka, Kyoto-pref.). Mean numbers of alleles per locus in the Yashima and Kameoka populations were 2.6 and 2.9, respectively, with corresponding mean observed heterozygosities of 0.36 and 0.52. These results based on the two additional populations confirm that our developed markers worked efficiently. The simple sequence repeat markers developed from expressed sequence tags in the present study should, therefore, be useful for explicating the population genetic structure of M. dorsalis and for future paternal analyses.

Abalone are one of the few marine taxa where aquaculture production dominates the global market as a result of increasing demand and declining natural stocks from overexploitation and disease. To better understand abalone biology, aid in conservation efforts for endangered abalone species, and gain insight into sustainable aquaculture, we created a draft genome of the red abalone (Haliotis rufescens). The approach to this genome draft included initial assembly using raw Illumina and PacBio sequencing data with MaSuRCA, before scaffolding using sequencing data generated from Chicago library preparations with HiRise2. This assembly approach resulted in 8,371 scaffolds and total length of 1.498 Gb; the N50 was 1.895 Mb, and the longest scaffold was 13.2 Mb. Gene models were predicted, using MAKER2, from RNA-Seq data and all related expressed sequence tags and proteins from NCBI; this resulted in 57,785 genes with an average length of 8,255 bp. In addition, single nucleotide polymorphisms were called on Illumina short-sequencing reads from five other eastern Pacific abalone species: the green (H. fulgens), pink (H. corrugata), pinto (H. kamtschatkana), black (H. cracherodii), and white (H. sorenseni) abalone. Phylogenetic relationships largely follow patterns detected by previous studies based on 1,784,991 high-quality single nucleotide polymorphisms. Among the six abalone species examined, the endangered white abalone appears to harbor the lowest levels of heterozygosity. This draft genome assembly and the sequencing data provide a foundation for genome-enabled aquaculture improvement for red abalone, and for genome-guided conservation efforts for the other five species and, in particular, for the endangered white and black abalone.