Peroxisomal KAT2 (3-ketoacyl-CoA thiolase 2) gene has a key role in gingerol biosynthesis in ginger (Zingiber officinale Rosc.).

Ginger is an important spice crop with medicinal values and gingerols are the most abundant pungent polyphenols present in ginger, responsible for most of its pharmacological properties. The present study focuses on the molecular mechanism of gingerol biosynthesis in ginger using transcriptome analysis. Suppression Subtractive Hybridization (SSH) was done in leaf and rhizome tissues using high gingerol-producing ginger somaclone B3 as the tester and parent cultivar Maran as the driver and generated high-quality leaf and rhizome Expressed Sequence Tags (ESTs). The Blast2GO annotations of the ESTs revealed the involvement of leaf ESTs in secondary metabolite production, identifying the peroxisomal KAT2 gene (Leaf EST 9) for the high gingerol production in ginger. Rhizome ESTs mostly coded for DNA metabolic processes and differential genes for high gingerol production were not observed in rhizomes. In the qRT-PCR analysis, somaclone B3 had shown high chalcone synthase (CHS: rate-limiting gene in gingerol biosynthetic pathway) activity (0.54 fold) in the leaves of rhizome sprouts. The presence of a high gingerol gene in leaf ESTs and high expression of CHS in leaves presumed that the site of synthesis of gingerols in ginger is the leaves. A modified pathway for gingerol/polyketide backbone formation has been constructed explaining the involvement of KAT gene isoforms KAT2 and KAT5 in gingerol/flavonoid biosynthesis, specifically the KAT2 gene which is otherwise thought to be involved mainly in ß-oxidation. The results of the present investigations have the potential of utilizing KAT/thiolase superfamily enzymes for protein/metabolic pathway engineering in ginger for large-scale production of gingerols. Supplementary Information: The online version contains supplementary material available at 10.1007/s13562-022-00825-x.

Development of EST-based methylation specific PCR (MSP) markers in Crocus sativus.

BACKGROUND: Saffron (Crocus sativus) is high valued spice crop, but due to its sterile nature, the crop is propagated exclusively through corms. Thus, the genetic base of this crop is very narrow, however, frequency of phenotypic variability is observed; and suggested the potential role of epigenetics in saffron crop growth and development. METHODS AND RESULTS: To facilitate epigenetic studies in saffron, we developed 1525 methylation-specific PCR (MSP) markers using MethPrimer. For this purpose, we used 6767 EST sequences of saffron available on the NCBI database. We also mine CpG islands (2555) and found that 32.7% of EST sequences had CpG islands. Out of 1525 MSP markers developed during the present study, 725 covered the CpG islands and 800 were without CpG islands. PCR amplification was found successful for 82% of MSP markers. A preliminary analysis suggested that 53.7% of genomic sites were methylated and more prominent (60% methylations) in non-CpG island regions, although, more comprehensive studies are required to validate it further. CONCLUSIONS: The epigenetic resource developed during the present study will strengthen the epigenetic studies like epiQTL mapping, epiGWAS to explore the molecular mechanisms and genomic/epigenomic regions associated with phenotype; and further may be utilized for saffron improvement programs through epibreeding.

Association mapping for abiotic stress tolerance using heat- and drought-related syntenic markers in okra.

BACKGROUND: Considerable production losses are caused by heat and drought stress in okra. Germplasm evaluation at genetic level is essential for the selection of promising genotypes. Lack of genomic information of okra limits the use of genetic markers. However, syntenic markers of some related family could be used for molecular characterization of major economic traits. METHODS AND RESULTS: Herein, 56 okra genotypes were evaluated for drought and heat tolerance. Sixty-one expressed sequence tags (ESTs) identified for heat and drought tolerance in cotton were searched from literature surveys and databases. The identified ESTs were BLAST searched into okra unigene database. Primers of selected okra unigenes were synthesized and amplified in all genotypes using standard polymerase chain reaction (PCR) protocol. Marker trait association (MTA) of the syntenic unigenes were identified between genotypic and phenotypic data on the basis of linkage disequilibrium Functional syntenic analysis revealed that out of these 61 cotton ESTs 55 had functional homology with okra unigenes. These 55 unigenes were used as markers for further analysis (amplification). Okra genotypes showed significance variations for all the physo-morphological parameters under heat and drought stress. Genotypes Perbhani Karanti, IQRA-III, Selection Super Green, Anmol and Line Bourd performed better under drought stress whereas genotypes Perbhani Karanti, IQRA-III, Green Gold, OK-1501 and Selection Super Green showed heat tolerance. Fifty markers showed amplification in okra. Fifty-six okra genotypes were clustered into three distinct populations. LD analysis has shown most significant linkage between markers Unigene43786 and Unigene3662. MTAs using MLM and GLM models revealed that 23 markers have significant associations (p < 0.05) with different traits under control and stressed conditions. Relative water content is associated with four markers (Unigene10673, Unigene99547, Unigene152901, and Unigene129684) under drought conditions. Whereas, Electrolyte leakage was associated with 3 markers (Unigene109922, Unigene28667 and Unigene146907) under heat stress. CONCLUSION: These identified unigenes may be helpful in the development of drought and heat tolerant genotypes in okra.

A systems biology study unveils the association between a melatonin biosynthesis gene, O-methyl transferase 1 (OMT1) and wheat (Triticum aestivum L.) combined drought and salinity stress tolerance.

MAIN CONCLUSION: Enhanced levels of endogenous melatonin in the root of wheat, mainly through the OMT1 gene, augment the antioxidant system, reestablish redox homeostasis and are associated with combined stress tolerance. A systems biology approach, including a collection of computational analyses and experimental assays, led us to uncover some aspects of a poorly understood phenomenon, namely wheat (Triticum aestivum L.) combined drought and salinity stress tolerance. Accordingly, a cross-study comparison of stress experiments was performed via a meta-analysis of Expressed Sequence Tags (ESTs) data from wheat roots to uncover the overlapping gene network of drought and salinity stresses. Identified differentially expressed genes were functionally annotated by gene ontology enrichment analysis and gene network analysis. Among those genes, O-methyl transferase 1 (OMT1) was highlighted as a more important (hub) gene in the dual-stress response gene network. Afterwards, the potential roles of OMT1 in mediating physiochemical indicators of stress tolerance were investigated in two wheat genotypes differing in abiotic stress tolerance. Regression analysis and correspondence analysis (CA) confirmed that the expression profiles of the OMT1 gene and variations in melatonin content, antioxidant enzyme activities, proline accumulation, H2O2 and malondialdehyde (MDA) contents are significantly associated with combined stress tolerance. These results reveal that the OMT1 gene may contribute to wheat combined drought and salinity stress tolerance through augmenting the antioxidant system and re-establishing redox homeostasis, probably via the regulation of melatonin biosynthesis as a master regulator molecule. Our findings provide new insights into the roles of melatonin in wheat combined drought and salinity stress tolerance and suggest a novel plausible regulatory node through the OMT1 gene to improve multiple-stress tolerant crops.

Data from expressed sequence tags from the organs and embryos of parthenogenetic Haemaphysalis longicornis.

OBJECTIVES: Haemaphysalis longicornis is the most important tick species in Japan and has a wide range of vector capacity. Due to its veterinary and medical importance, this tick species has been used as a model for tick/vector biological studies. To identify the key molecules associated with physiological processes during blood feeding and embryogenesis, full-length cDNA libraries were constructed using the fat body, hemocytes-containing hemolymph, midgut, ovary and salivary glands of fed females and embryos of the laboratory colony of parthenogenetic H. longicornis. The sequences of cDNA from the salivary glands had been already released. However, the related information is still poor, and the other expressed sequence tags have not yet been deposited. DATA DESCRIPTION: A total of 39,113 expressed sequence tags were obtained and deposited at the DNA DataBank of Japan. There were 7745 sequences from embryos, 7385 from the fat body, 8303 from the hemolymph including hemocytes, 7385 from the midgut, and 8295 from the ovary. The data, including expressed sequence tags from the salivary glands was summarized into Microsoft Excel files. Sharing this data resource with the tick research community will be valuable for the identification of novel genes and advance the progress of tick research.

Preliminary investigations on the pathogenesis-related protein expression profile of the medicinal herb Macleaya cordata and anti-bacterial properties of recombinant proteins.

The plant pathogenesis-related (PR) proteins play a crucial role in the defense of plants against pathogens and orchestrate the innate immune system of plants. In this paper, a non-normalized cDNA library of the leaf was constructed to obtain a comprehensive view of PR proteins of Macleaya cordata. Specifically, 511 expressed sequence tags (ESTs) were generated using Sanger sequencing. All ESTs were assembled into 364 non-redundancy sequences, including 78 clusters and 286 singlets. The PR protein expression profile of the medicinal herb M. cordata has been investigated and is represented by defensin, lipid-transfer protein, (S)-norcoclaurine synthase, and major allergen protein, suggesting that the herb contains rich active proteins against pathogens. Furthermore, two defensins were selected for recombinant expression in yeast, and the antimicrobial activities were explored. Since they both present a broad antimicrobial spectrum, they are of particular importance for agricultural and medicinal applications. Our study describes defensins in Papaveraceae for the first time and provides novel insights into the effective components. In addition to the alkaloids, PR proteins (such as defensins, lipid transfer proteins, (S) - norcoclaurine synthase, major allergen protein, and Class IV chitinases) are involved in the antibacterial and anti-inflammatory activities of M. cordata.

Identification of putative miRNAs from expressed sequence tags of Gnetum gnemon L. and their cross-kingdom targets.

Wild edible plants are often found to be rich sources of nutrients and medicinally beneficial compounds with pharmacological activities. Gnetum gnemon is a nutritionally important plant and a popular food source in parts of Assam and North-East India. Various microRNAs (miRNAs) have been recently identified in many plants; however, there are no records of identification of miRNAs in any species of Gnetum. The prediction of miRNA-target associations in G. gnemon is an important step to facilitate functional genomics studies in this species. In the present study, all known miRNAs from plants available in public domain were used to search for the conserved G. gnemon miRNA homologues in publicly accessible expressed sequence tags (ESTs) in NCBI database. An aggregate of 20 new potential miRNAs belonging to two diverse miRNA families (miR399 and miR5021) were identified through a homology-based search by following stringent filtering criteria. To investigate the potential cross-kingdom effects of the identified miRNAs, we further identified the putative target genes of G. gnemon miRNAs in human transcriptome and analyzed them against the NCBI non-redundant protein database. The KEGG analysis of the target genes indicated that these genes were involved in different metabolic pathways such as caffeine metabolism, drug metabolism, and nitrotoluene degradation. The target genes of G. gnemon miRNAs in humans were found to be associated with various disorders of both hereditary and non-hereditary origin. These results could help to shed new light on understanding of miRNA-mRNAs functional networks in this species and its potential use as a small RNA-based therapy against some human diseases.

Identification and molecular analysis of cashmere goat lncRNAs reveal their integrated regulatory network and potential roles in secondary hair follicle.

Long non-coding RNAs (lncRNAs) is a class of eukaryotic transcripts with length of more than 200 bp. They contribute to the regulation of gene expressions involved in multiple processes including the skin cell proliferation, differentiation, and reconstruction of the secondary hair follicles (SHFs). In this study, firstly, we identified 16 putative lncRNAs from SHFs of cashmere goat based on the EST sequences from NCBI database. Secondly, we investigated their transcriptional pattern in SHFs of cashmere goat, and constructed their ceRNA regulatory networks. The RT-qPCR results showed four lncRNAs (lncRNA-475074, -052149, -052140, and -051789) were significantly up-regulated, and nine lncRNAs (lncRNA-711032, -475083, -475070, -052139, -052127, -052037, -051903, -051847, and -051804) were significantly down-regulatd in anagen SHFs of cashmere goat. CeRNA networks analysis revealed complex interactional relationship among lncRNAs, miRNAs and mRNAs. Further, the KEGG pathway enrichment was performed for the potential target genes of the identified lncRNAs based on bioinformatics technique, and the results indicated that differentially expressed lncRNAs directly or indirectly might regulate potential target genes. Our results from this study will provide a significant information for further exploring the functions and possible mechanisms of the identified lncRNAs in SHFs of cashmere goat.

Development of RNA-seq-based molecular markers for characterizing Thinopyrum bessarabicum and Secale introgressions in wheat.

Sequence-based markers have added a new dimension in the efficiency of identifying alien introgressions in wheat. Expressed sequence tag-sequence tagged sites (EST-STS) markers have proved useful in tracing alien chromatin. In this study, we report the development of Thinopyrum bessarabicum- and Secale anatolicum-specific EST-STS markers and their application in tracing respective alien chromatin introgressions in wheat. The parental lines, Chinese Spring (CS), ISR991.1 (CS/Th. bessarabicum amphidiploid), and ISR1049.2 (CS/Secale anatolicum amphidiploid), were used as core experimental materials. Using comparative analysis of RNA-Seq data, 10 903 and 10 660 candidate sequences specific to Th. bessarabicum and S. anatolicum, respectively, were assembled and identified. To validate the genome specificity of these candidate sequences, 68 and 64 EST-STS markers were developed from randomly selected candidate sequences of Th. bessarabicum and S. anatolicum, respectively, and tested on sets of alien addition lines. Fifty-five and 53 markers for Th. bessarabicum and S. anatolicum chromatin, respectively, were assigned to chromosomal location(s), covering all seven chromosomes. Approximately 83% of S. anatolicum-specific markers were transferable to S. cereale. The genome-specific candidate sequences identified and the EST-STS markers developed will be valuable resources for exploitation of Th. bessarabicum and Secale species diversity in wheat and triticale breeding.

Mining of miRNAs from EST data in Dendrobium nobile.

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.

Whole-Transcriptome Analysis Unveils the Synchronized Activities of Genes for Fructans in Developing Tubers of the Jerusalem Artichoke.

Helianthus tuberosus L., known as the Jerusalem artichoke, is a hexaploid plant species, adapted to low-nutrient soils, that accumulates high levels of inulin in its tubers. Inulin is a fructose-based polysaccharide used either as dietary fiber or for the production of bioethanol. Key enzymes involved in inulin biosynthesis are well known. However, the gene networks underpinning tuber development and inulin accumulation in H. tuberous remain elusive. To fill this gap, we selected 6,365 expressed sequence tags (ESTs) from an H. tuberosus library to set up a microarray platform and record their expression across three tuber developmental stages, when rhizomes start enlarging (T0), at maximum tuber elongation rate (T3), and at tuber physiological maturity (Tm), in "VR" and "K8-HS142"clones. The former was selected as an early tuberizing and the latter as a late-tuberizing clone. We quantified inulin and starch levels, and qRT-PCR confirmed the expression of critical genes accounting for inulin biosynthesis. The microarray analysis revealed that the differences in morphological and physiological traits between tubers of the two clones are genetically determined since T0 and that is relatively low the number of differentially expressed ESTs across the stages shared between the clones (93). The expression of ESTs for sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT), the two critical genes for fructans polymerization, resulted to be temporarily synchronized and mirror the progress of inulin accumulation and stretching. The expression of ESTs for starch biosynthesis was insignificant throughout the developmental stages of the clones in line with the negligible level of starch into their mature tubers, where inulin was the dominant polysaccharide. Overall, our study disclosed candidate genes underpinning the development and storage of carbohydrates in the tubers of two H. tuberosus clones. A model according to which the steady-state levels of 1-SST and 1-FFT transcripts are developmentally controlled and might represent a limiting factor for inulin accumulation has been provided. Our finding may have significant repercussions for breeding clones with improved levels of inulin for food and chemical industry.

Identification of miR-802-5p and its involvement in type 2 diabetes mellitus.

MicroRNAs (miRNA) are recently discovered endogenous, small noncoding RNAs (of 22 nucleotides) that play pivotal roles in gene regulation. They are involved in post-transcriptional control of gene expression. miRNAs are emerging as important regulators of cell proliferation, development, cancer formation, stress responses, cell death and physiological conditions. Increasing evidence has demonstrated the human miRNAs bind to their target mRNA sequences with perfect or near-perfect sequence complementarily. This provides a powerful strategy for discovering potential type 2 diabetes mellitus (T2DM) targets and gives the probability to exploit them for diagnostic and therapeutic causes. About 6% of the world population is affected by T2DM, and it is recognized as a global epidemic by the World Health Organization. At present there is no valid biomarker to control or manage T2DM. Therefore, the present study applied a mature sequence of miRNAs from publicly accessible databases to identify the miRNA from T2DM expressed sequence tags, and the results are detailed and discussed below.

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately -7°C) and cold-acclimated (CA; leaf LT50 approximately -50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Screening of broad spectrum natural pesticides against conserved target arginine kinase in cotton pests by molecular modeling.

Cotton is an economically important crop and its production is challenged by the diversity of pests and related insecticide resistance. Identification of the conserved target across the cotton pest will help to design broad spectrum insecticide. In this study, we have identified conserved sequences by Expressed Sequence Tag profiling from three cotton pests namely Aphis gossypii, Helicoverpa armigera, and Spodoptera exigua. One target protein arginine kinase having a key role in insect physiology and energy metabolism was studied further using homology modeling, virtual screening, molecular docking, and molecular dynamics simulation to identify potential biopesticide compounds from the Zinc natural database. We have identified four compounds having excellent inhibitor potential against the identified broad spectrum target which are highly specific to invertebrates.

Genetic diversity and population structure analyses of Plectranthus edulis (Vatke) Agnew collections from diverse agro-ecologies in Ethiopia using newly developed EST-SSRs marker system.

BACKGROUND: Plectranthus edulis (Vatke) Agnew (locally known as Ethiopian dinich or Ethiopian potato) is one of the most economically important edible tuber crops indigenous to Ethiopia. Evaluating the extent of genetic diversity within and among populations is one of the first and most important steps in breeding and conservation measures. Hence, this study was aimed at evaluating the genetic diversity and population structure of this crop using collections from diverse agro-ecologies in Ethiopia. RESULTS: Twenty polymorphic expressed sequence tag based simple sequence repeat (EST-SSRs) markers were developed for P. edulis based on EST sequences of P. barbatus deposited in the GenBank. These markers were used for genetic diversity analyses of 287 individual plants representing 12 populations, and a total of 128 alleles were identified across the entire loci and populations. Different parameters were used to estimate the genetic diversity within populations; and gene diversity index (GD) ranged from 0.31 to 0.39 with overall mean of 0.35. Hierarchical analysis of molecular variance (AMOVA) showed significant but low population differentiation with only 3% of the total variation accounted for variation among populations. Likewise, cluster and STRUCTURE analyses did not group the populations into sharply distinct clusters, which could be attributed to historical and contemporary gene flow and the reproductive biology of the crop. CONCLUSIONS: These newly developed EST-SSR markers are highly polymorphic within P. edulis and hence are valuable genetic tools that can be used to evaluate the extent of genetic diversity and population structure of not only P. edulis but also various other species within the Lamiaceae family. Among the 12 populations studied, populations collected from Wenbera, Awi and Wolaita showed a higher genetic diversity as compared to other populations, and hence these areas can be considered as hot spots for in-situ conservation as well as for identification of genotypes that can be used in breeding programs.

Ovine keratome: identification, localisation and genomic organisation of keratin and keratin-associated proteins.

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin-associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST-derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.

Characterization of leaf transcriptome, development and utilization of unigenes-derived microsatellite markers in sugarcane (Saccharum sp. hybrid).

Sugarcane (Saccharum species hybrid) is the major source of sugar (> 80% sugar) in the world and is cultivated in more than 115 countries. It has recently gained attention as a source of biofuel (ethanol). Due to genomic complexity, the development of new genomic resources is imperative in understanding the gene regulation and function, and to fine tune the genetic improvement of sugarcane. In this study, a cDNA library was constructed from mature leaves so as to develop ESTs resources which were further compared with nucleotide and protein databases to explore the functional identity of sugarcane genes. The non-redundant ESTs (unigenes) were categorized into 18 metabolic functions. The major categories were bioenergetics and photosynthesis (4%), cell metabolism (5%), development related protein (3%), membrane-related, mobile genetic elements (5%), signal transduction (2%), DNA (1%), RNA (1%) and protein (2%) metabolism, other metabolic processes (3%), transcription factors (1%), transport (4%) and proteins related to stress/defense (4%). From 540 unique ESTs, 212 simple sequence repeats were identified, of which 206 were from 463 singlets and six were mined from 77 contig sequences. A total of 540 unique EST sequences were used for SSR search of which 97 (17.9%) contained specified SSR motifs, generating 212 unique SSRs. The genes characterized in this study and the EST-derived microsatellite markers identified from the cDNA library will enrich genomic resources for association- and linkage-mapping studies in sugarcane.

Analysis of population structure and genetic diversity in an exotic germplasm collection of Eleusine coracana (L.) Gaertn. using genic-SSR markers.

Finger millet (Eleusine coracana (L.) Geartn.) is one of the important small millets serves as a food security crop because of its high nutritional values. The complex tetraploid genome of finger millet requires a large number of informative, functional DNA markers for different applications in genetics and breeding. Yet, less number of simple sequence repeat (SSR) markers have been developed from expressed sequence tags in finger millet. In the present study, 56 new genic SSR markers were developed from publicly available drought related ESTs. The 43 polymorphic markers were used to evaluate polymorphism, revealed a range of PIC value 0.41 to 0.79. Our results suggest that, analyzed genotypes have high genetic diversity with an average gene diversity (h) of 0.176 and Shannon's information index (I) of 0.315. We conclude that there was a higher gene exchange within populations, by the value of overall gene flow (Nm) of 0.7721. The unweighted pair group method with arithmetic mean and neighbor joining dendrogram generated three main clusters to differentiate genotypes and these results were also confirmed by PCA and PCoA analysis. The high genetic diversity (77%) was found within the populations in the analysis of molecular variance. A Bayesian model-based cluster analysis evidenced a high extent of admixture between the gene pools from the different geographical origins. Population based cluster analyses pointed out a strong pattern of 'isolation by distance'. Overall, these results underscored that this study showed a significantly high level of polymorphism, adequate genetic diversity and population structure which expand the modern genetic resources and its utility in various applications in genetics and genomics including association mapping and breeding.

Generation and characterization of expressed sequence tags (ESTs) from coralloid root cDNA library of Cycas debaoensis.

A normalized full-length cDNA library was constructed from the coralloid roots of Cycas debaoensis by the DSN (duplex-specific nuclease) normalization method combined with the SMART (Switching Mechanism At 5' end of the RNA Transcript) technique. The titer of the original cDNA library was about 1.5 × 106 cfu·mL-1 and the average insertion size was about 1 kb with a high recombination rate (97%). The 5011 high-quality expressed sequence tags (ESTs) were obtained from 5393 randomly picked cDNA clones. Clustering and assembly of ESTs resulted in 2984 unique sequences, consisting of 618 contigs and 2366 singlets. EST sequence annotation revealed that 2333 and 1901 unigenes were functionally annotated in the NCBI non-redundant database and Swiss-Prot protein database, respectively. Functional analysis demonstrated that 1495 (50.1%) unigenes were associated with 4082 Gene Ontology (GO) terms. A total of 847 unigenes were grouped into 22 Cluster of Orthologous Groups (COG) functional categories. Based on the EST dataset, 22 ESTs that encoded putative receptor-like protein kinase (RLK) genes were screened. Furthermore, a total of 94 simple sequence repeats (SSRs) were discovered, of which 20 loci were successfully amplified in C. debaoensis. This study is the first EST analysis for the coralloid roots of C. debaoensis and provides a valuable genomic resource for novel gene discovery, gene expression and comparative genomics, conservation and management studies as well as applications in C. debaoensis and related cycad species.

Analysis of Expressed Sequence Tags (EST) in Date Palm.

Expressed sequence tags (EST) were generated from a normalized cDNA library of the date palm Sukkari cv. to understand the high-quality and better field performance of this well-known commercial cultivar. A total of 6943 high-quality ESTs were generated, out of them 6671 are submitted to the GenBank dbEST (LIBEST_028537). The generated ESTs were assembled into 6362 unigenes, consisting of 494 (14.4%) contigs and 5868 (84.53%) singletons. The functional annotation shows that the majority of the ESTs are associated with binding (44%), catalytic (40%), transporter (5%), and structural molecular (5%) activities. The blastx results show that 73% of unigenes are significantly similar to known plant genes and 27% are novel. The latter could be of particular interest in date palm genetic studies. Further analysis shows that some ESTs are categorized as stress/defense- and fruit development-related genes. These newly generated ESTs could significantly enhance date palm EST databases in the public domain and are available to scientists and researchers across the globe. This knowledge will facilitate the discovery of candidate genes that govern important developmental and agronomical traits in date palm. It will provide important resources for developing genetic tools, comparative genomics, and genome evolution among date palm cultivars.