Expressed Sequence Tags Sequences Research Articles

Development and Validation of Gene-Based SSR Markers in the Genus Mesembryanthemum.

Bioinformatics tools have been employed for the direct development of gene-based simple sequence repeat (SSR) markers. Through the analysis of 28,056 Mesembryanthemum expressed sequence tag (EST) sequences, a total of 5,851 ESTs containing SSRs were identified, amounting to approximately 17.07 Mb. Among these, 938 EST sequences harbored more than one SSR marker, and 788 EST-SSR sequences were found in compound form. The most prevalent types of SSR motifs were mononucleotide repeats (MNRs), accounting for 44%, followed by di-nucleotide repeats (DNRs) at 37%, and trinucleotide repeats (TNRs) at 16%. Notably, TNR or longer SSR motifs primarily consisted of shorter repeat lengths, with only 51 motifs containing 10 or more repeats. The BLASTX analysis successfully assigned functions to 4,623 (79%) of the EST sequences. Among the developed primer sets, 21 primers amplified a total of 65 alleles, with primer PMA79 EST-SSR exhibiting the maximum of six alleles. The polymorphic information content (PIC) values ranged from 0 to 0.76, with a mean of 0.47. The marker index (MI) and discriminating power (D) values reached 0.66 (primer PMA63) and 0.95 (primer PMA20), respectively. Utilizing the unweighted pair group method with arithmetic mean (UPGMA), a dendrogram was constructed, successfully segregating the 24 Mesembryanthemum genotypes into three distinct clusters, with a similarity coefficient ranging from 0.96 to 0.38. In this study, we have developed a total of 83 EST-SSR primer pairs specific to the Mesembryanthemum genus. These newly developed EST-SSRs will serve as valuable tools for researchers, particularly molecular breeders, enabling gene-based identification and trait selection through marker-assisted breeding approaches.

Analysis of express sequence tags from Arbuscular Mycorrhizal fungi (AMF) express during the pre-symbiotic stage of the fungus

Arbuscular Mycorrhizal Fungi (AMF) are symbiotically associated with the roots of higher plants which enhance the overall nutritional status of plants. It is known that the overall growth and development of plants not only depend upon the performance of specific genotypes but their association is also affected by the various types of microbes. Therefore, the major aim of the study was the isolation and characterization of gene expression during the presymbiotic stage of AMF. In the present study, total RNA was extracted from spores of Rhizophagus irregularis grown under root organ culture (ROC). cDNA library was prepared and expressed sequence tag (EST) analysis was performed. Approximately 150 random clones were selected for EST sequencing analysis. Out of the 150 clones obtained, 1.5% carried inserts of the rRNA gene cluster. After excision, inserts from 72 randomly selected clones were sequenced. Database searches revealed that 74% of the clones had similarities to already known sequences. Putative genes for Ca2+-regulated symbiotic genes, cell cycle regulatory factors and precursors for lipid biosynthesis genes were discovered. A full-length mating gene was discovered using rapid amplification of cDNA ends (RACE). Studies revealed the presence of a putative mating type gene in mycorrhizal species that are evident in the sexual origin of AMF.

In silico identification of novel and conserved microRNAs and targets in peppermint (Mentha piperita) using expressed sequence tags (ESTs)

ObjectivesMentha piperita, generally known as peppermint is a popular herb cultivated in the temperate region around the world belongs to the family Lamiaceae. M. piperita is widely used as traditional food flavoring, in addition they are as well used to treat cold, fever and throat inflammation, and they have anti-viral and anti-fungal properties. The characteristics of MicroRNAs (miRNAs) includes, small size with single stranded non-coding regions having 22 nucleotides. They are evolutionary conserved and actively involved in post-transcriptional regulation of RNAs, which plays various important roles in metabolic and cellular processes of animals and plants. MethodsThe present study reports the potent computational method based on homology search used to find potential miRNA of peppermint. EST sequences were retrieved from NCBI GenBank and BLAST against with plant miRNAs. By various filtration criteria we identified potential miRNAs from miRNA family with suitable properties. Results and ConclusionA total of 1216 ESTs were and 1188 unigenes were identified in this study. A set of prospective target genes were identified in peppermint based on sequence homologies mainly encodes for transcription factor, transport inhibitor like protein, GRR1-like protein and DNA-binding protein. The present study endows valuable information on miRNA in peppermint which will accelerate further research in peppermint.

In Silico Mining and Characterization of High-Quality SNP/Indels in Some Agro-Economically Important Species Belonging to the Family Euphorbiaceae.

(1) Background: To assess the genetic makeup among the agro-economically important members of Euphorbiaceae, the present study was conducted to identify and characterize high-quality single-nucleotide polymorphism (SNP) markers and their comparative distribution in exonic and intronic regions from the publicly available expressed sequence tags (ESTs). (2) Methods: Quality sequences obtained after pre-processing by an EG assembler were assembled into contigs using the CAP3 program at 95% identity; the mining of SNP was performed by QualitySNP; GENSCAN (standalone) was used for detecting the distribution of SNPs in the exonic and intronic regions. (3) Results: A total of 25,432 potential SNPs (pSNP) and 14,351 high-quality SNPs (qSNP), including 2276 indels, were detected from 260,479 EST sequences. The ratio of quality SNP to potential SNP ranged from 0.22 to 0.75. A higher frequency of transitions and transversions was observed more in the exonic than the intronic region, while indels were present more in the intronic region. C↔T (transition) was the most dominant nucleotide substitution, while in transversion, A↔T was the dominant nucleotide substitution, and in indel, A/- was dominant. (4) Conclusions: Detected SNP markers may be useful for linkage mapping; marker-assisted breeding; studying genetic diversity; mapping important phenotypic traits, such as adaptation or oil production; or disease resistance by targeting and screening mutations in important genes.

In-Silico Mining and Characterization of EST-SSRs for the Genetic Diversity Analysis of Lemon

One of the economically important plants of the family of Rutaceae, Citrus limon L. Osbeck also known as lemon exists as different varieties of which many are indigenous to our country. Therefore, to assess the diversity or the genetic variations among the lemon varieties, the development of molecular markers is necessitated. Expressed Sequence Tags (ESTs) are small region of expressed DNA sequences of a genome which are highly conserved across species and are co-dominant in nature by having Simple Sequence Repeats (SSRs) unlike RAPD and AFLP which are dominant. Hence, this study was taken up for mining and characterizing SSR markers from the publically available EST data. The ESTs downloaded from NCBI database were mined for SSRs using MISA followed by primer designing using Primer3. Functional domains and GO analysis were performed on Blast2GO and primer-BLAST was done to check the suitability of the primers flanking the SSR regions. Total 1505 sequences were retrieved from NCBI database which after initial clean up filtered 1406 sequences containing 59 contigs and 1347 singletons. Total 80 SSRs were identified with frequency of 1SSR/Kb to be 8.27 of the total sequences examined. The 76 SSR containing EST sequences were functionally annotated, which resulted in 20 annotated ESTs and 22 ESTs having IPR (PFAM) domains. Successful primer pairs were designed for these EST-SSRs and out of these, 33 sequences having >200bp products were sorted of which 26 were optimised further and finally 12 primer pairs gave successful primer-BLAST results without any mismatch with two other citrus species. Thus, the findings gave an insight on the distribution and frequency of SSR in the ESTs of lemon. In the future, the usage of these functional molecular markers may further assist to explore the genetic diversity analysis of different lemon varieties.

Analysis of Genetic Diversity in Wild and Domesticated Stocks of the Tropical Abalone Haliotis asinina by Microsatellite Polymorphism

The basic information on genetic diversity of wild and hatchery-propagated stocks of tropical abalone, Haliotis asinina is important for the construction of a breeding scheme leading to the sustainable culturing activity of this species. In this study, 2,876 expressed sequence tags (ESTs) were in silico analyzed and 178 EST sequences contained microsatellite motifs. Four loci (DW455, DW503, PHe177, and PT102) of type I and two loci (Haμ9 and Haμ10) of type II microsatellites were applied for genetic diversity studies. The mean number of alleles per locus and observed heterogeneity in wild populations were 4.167 and 0.483, and 4.833 and 0.528 for CAME (east coast) and TRGW (west coast) while those of hatchery-propagated samples were 3.000 and 0.708, 4.500 and 0.479, 5.167 and 0.524, and 6.333 and 0.527 for PHIH (2nd generation, G2; Philippines), SAMH (G1), SMaRT-TRGH (G8) and SMaRT-SICH (G8), respectively. The numbers of alleles, observed heterozygosity and effective population sizes suggested no severe reduction of genetic diversity in our breeding program of H. asinina. However, reduced Ne was observed in the cultured stocks from the Philippines (PHIH). FST-statistics and the exact test between pairs of samples revealed significant genetic differences between all pairwise comparisons of samples in this study.

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.

Application of Efficient Express Sequence Tags Information for Classification and Functional Study of Simple Sequence Repeats in Cattle Testis Tissue

Genomic markers play an important role in tracing the flow of genetic causality of observable signals in animals and plants. In farm animals, the participation of male animals in the gene pool of subsequent generations are much higher than female animals and testes are the most important organs of the male reproductive system. This study was conducted to investigate simple sequence repeats (SSR) within the expressed sequence tags (ESTs) in order to classify the Bos taurus testis tissue’s genes for their relationship and specificity with related reproductive domains. A total of 48,549 publicly available EST sequences from cattle testis tissue downloaded from GenBank database, out of which, 10,237 sequences that their library made from testis tissue were extracted and specialized as the studied sequences using several searching tools and software. Across these selective sequences, 2,039 contigs, 5,097 singletons, and 153 SSRs were detected. EST-SSRs were subsequently evaluated using GenBank and categorized based on their functions in biological systems of dairy cattle. Investigation of these motifs showed that the identified EST-SSRs can be classified into 48 types that GT in dinucleotides and GCC in trinucleotides had the highest frequency. Annotation and gene ontology analysis revealed a relationship among 54 domains with the observed SSRs. Localization and characterization of such markers can help tracing the production of amino acids coded by identified repeats as shown in this study.

Characterization of the Wheat-Psathyrostachys huashania Keng 2Ns/2D Substitution Line H139: A Novel Germplasm With Enhanced Resistance to Wheat Take-All.

Take-all is a devastating soil-borne disease that affects wheat production. The continuous generation of disease-resistance germplasm is an important aspect of the management of this pathogen. In this study, we characterized the wheat-Psathyrostachys huashania Keng (P. huashania)-derived progeny H139 that exhibits significantly improved resistance to wheat take-all disease compared with its susceptible parent 7182. Sequential genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (mc-FISH) analyses revealed that H139 is a stable wheat-P. huashania disomic substitution line lacking wheat chromosome 2D. Expressed sequence tag-sequence tagged site (EST-STS) marker and Wheat Axiom 660K Genotyping Array analysis further revealed that H139 was a novel wheat-P. huashania 2Ns/2D substitution line. In addition, the H139 line was shown to be cytologically stable with a dwarf phenotype and increased spikelet number. These results indicate that H139, with its enhanced wheat take-all disease resistance and desirable agronomic traits, provides valuable genetic resources for wheat chromosome engineering breeding.

Single-Nucleotide Polymorphisms (SNP) Mining and Their Effect on the Tridimensional Protein Structure Prediction in a Set of Immunity-Related Expressed Sequence Tags (EST) in Atlantic Salmon (Salmo salar).

Single-nucleotide polymorphisms (SNPs) are single genetic code variations considered one of the most common forms of nucleotide modifications. Such SNPs can be located in genes associated to immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections affecting the productive sector. In this study, a set of immune-related genes (cc motif chemokine 19 precursor [ccl19], integrin β2 (itβ2, also named cd18), glutathione transferase omega-1 [gsto-1], heat shock 70 KDa protein [hsp70], major histocompatibility complex class I [mhc-I]) were analyzed to identify SNPs by data mining. These genes were chosen based on their previously reported expression on infectious pancreatic necrosis virus (IPNV)-infected Atlantic salmon phenotype. The available EST sequences for these genes were obtained from the Unigene database. Twenty-eight SNPs were found in the genes evaluated and identified most of them as transition base changes. The effect of the SNPs located on the 5’-untranslated region (UTR) or 3’-UTR upon transcription factor binding sites and alternative splicing regulatory motifs was assessed and ranked with a low-medium predicted FASTSNP score risk. Synonymous SNPs were found on itβ2 (c.2275G > A), gsto-1 (c.558G > A), and hsp70 (c.1950C > T) with low FASTSNP predicted score risk. The difference in the relative synonymous codon usage (RSCU) value between the variant codons and the wild-type codon (ΔRSCU) showed one negative (hsp70 c.1950C > T) and two positive ΔRSCU values (itβ2 c.2275G > A; gsto-1 c.558G > A), suggesting that these synonymous SNPs (sSNPs) may be associated to differences in the local rate of elongation. Nonsynonymous SNPs (nsSNPs) in the gsto-1 translatable gene region were ranked, using SIFT and POLYPHEN web-tools, with the second highest (c.205A > G; c484T > C) and the highest (c.499T > C; c.769A > C) predicted score risk possible. Using homology modeling to predict the effect of these nonsynonymous SNPs, the most relevant nucleotide changes for gsto-1 were observed for the nsSNPs c.205A > G, c484T > C, and c.769A > C. Molecular dynamics was assessed to analyze if these GSTO-1 variants have significant differences in their conformational dynamics, suggesting these SNPs could have allosteric effects modulating its catalysis. Altogether, these results suggest that candidate SNPs identified may play a crucial potential role in the immune response of Atlantic salmon.

In silico identification of microRNAs and their target genes in watermelon (Citrullus lanatus)

Watermelon (Citrullus lanatus) is an annual herbaceous crop, mainly grown for its fleshy fruit, which is an excellent source of vitamins and therefore fetches a huge market demand all over the world. Yield of watermelon can be improved by increasing the productivity that relies on plant growth, development and stress response. MicroRNAs (miRNAs), a group of short (∼22 nt), noncoding, endogenous and evolutionary conserved post-transcriptional regulatory RNAs, play a major role in various biological and metabolic processes associated with growth, development and stress response. In this study, we deployed a comparative genomics-based approach using expressed sequence tags (ESTs) to identify and characterize potential miRNAs and their target genes in watermelon. Three potential miRNAs (cla-miR-2515, cla-miR482d and cla-miR-8461) were predicted from 17,594 publicly available watermelon EST sequences, according to a series of filtering criteria used in the protocol. Moreover, 21 potential mRNA targets encoding for 19 different proteins linked to various metabolic processes such as inositol-3-phosphate synthase activity, metal ion binding and oxidoreductase activity were identified in watermelon genome, based on perfect or nearly perfect complementarity of miRNAs with their targeted mRNA sequences. Phylogenetic analysis of these miRNAs revealed that cla-miR8461 and cla-miR2515 are unique miRNAs closely related to Schistosoma mansoni and Drosophila pseudoobscura, respectively.

Development and validation of whole genome-wide and genic microsatellite markers in oil palm (Elaeis guineensis Jacq.): First microsatellite database (OpSatdb)

The availability of large expressed sequence tag (EST) and whole genome databases of oil palm enabled the development of a data base of microsatellite markers. For this purpose, an EST database consisting of 40,979 EST sequences spanning 27 Mb and a chromosome-wise whole genome databases were downloaded. A total of 3,950 primer pairs were identified and developed from EST sequences. The tri and tetra nucleotide repeat motifs were most prevalent (each 24.75%) followed by di-nucleotide repeat motifs. Whole genome-wide analysis found a total of 245,654 SSR repeats across the 16 chromosomes of oil palm, of which 38,717 were compound microsatellite repeats. A web application, OpSatdb, the first microsatellite database of oil palm, was developed using the PHP and MySQL database (https://ssr.icar.gov.in/index.php). It is a simple and systematic web-based search engine for searching SSRs based on repeat motif type, repeat type, and primer details. High synteny was observed between oil palm and rice genomes. The mapping of ESTs having SSRs by Blast2GO resulted in the identification of 19.2% sequences with gene ontology (GO) annotations. Randomly, a set of ten genic SSRs and five genomic SSRs were used for validation and genetic diversity on 100 genotypes belonging to the world oil palm genetic resources. The grouping pattern was observed to be broadly in accordance with the geographical origin of the genotypes. The identified genic and genome-wide SSRs can be effectively useful for various genomic applications of oil palm, such as genetic diversity, linkage map construction, mapping of QTLs, marker-assisted selection, and comparative population studies.

Construction of a cDNA library and preliminary analysis of the expressed sequence tags of the earthworm Eiseniafetida (Savigny, 1826).

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 <1×10−8), of which 168 were annotated through Gene Ontology analysis. The STRING database was used to determine relationships among the 168 ESTs, identifying associated genes involved in protein-protein interactions and gene expression regulation. Based on nucleic acid and protein sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a high-quality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms.

Prediction and analysis of GH14 family β-amylases in oat seedling extract: Structure and function insights using in silico approaches

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.

Genetic analysis and molecular mapping of stripe rust resistance in an excellent wheat line Sanshumai1

Stripe rust, caused by Puccinia striiformis Westend. f. sp. tritici (Pst), is one of the most widespread diseases in wheat (Triticum aestivum L.). Planting resistant cultivars has been constantly practiced for decades as the best strategy to defense against the variations of prevalent Pst races. The wheat line Sanshumai1 was developed from an interspecific hybridization of Haynaldia villosa (L) Schur. (2n = 2× = 14, VV)/Triticum turgidum L. var. durum (4n = 4× = 28, AABB)///Yanxiaomai. Sanshumai1 has all-stage resistance to most of known stripe rust races in China, including three widely virulent races CYR31, CYR32, and CYR33. To identify stripe rust resistance gene in this line, Sanshumai1 was crossed with the susceptible genotype, Mingxian169, and the F1, F2, F3, and BC1 generations were inoculated with Pst races under the controlled greenhouse conditions. The genetic results indicated that two stripe rust resistance genes in Sanshumai1, temporarily designated as YrS1 and YrS2, confer resistance to CYR31 and Su11–11, respectively. Using bulked segregant analysis (BSA) methodology, we identified five simple sequence repeat (SSR) markers and two expressed sequence tag-sequence tagged site (EST-STS) markers associated with YrS1 on the short arm of chromosome 3D. The genetic distances of the two closest flanking markers, namely Xcfd79 and Xwmc674, were 4.1 and 8.7 centiMorgans, respectively. In addition, we identified four SSR markers associated with YrS2 on the long arm of chromosome 4D. The genetic distances of the two closest flanking markers, namely Xcfd84 and Xgwm194, were 6.8 and 7.1 centiMorgans, respectively. Based on the chromosomal location, reaction patterns, and pedigree analysis, these two genes are likely novel resistance genes. These two genes and the flanking markers developed from this study are expected to be useful in pyramiding YrS1 and YrS2 with other Yr genes to develop wheat cultivars with high-level and durable resistance to stripe rust and may also benefit marker assisted selection (MAS) in breeding programs.

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.

Identification, expression, and methylation of miR-7110 and its involvement in type 1 diabetes mellitus

MicroRNAs are a novel group of universally present non-coding RNA that has been implicated in the wide-ranging biological process and are critical in the manifestation of diverse disease including diabetes mellitus (DM). Although there are so many numbers of micro RNAs involved in DM none of them effective for the suppression of Type I DM (T1DM), hence there is an urgent need for identifying exact microRNAs that may serve as a candidate biomarker for T1DM. Moreover, we know that expressed sequence tags (ESTs) are random single pass sequences reads which displays the condition/tissue-specific transcribes of an organism. In the present study, we have applied bioinformatics tools to identify hsa-mir-7110-5p from diabetes mellitus using EST resources. Our studies revealed that hsa-mi-7110 was confirmed in the T1DM specific EST sequence among the predicted miRNA secondary structure. It has been known that miRNAs are known to be an important factor involved in the initiation and progression of T1DM. Based on the above information, we also deciphered the molecular mechanism and the role of miR-7110 from T1DM patients and found that miR-7110 was a novel prognostic, diagnostic and therapeutic target for T1DM.

Expressed sequence tags (ESTs)–based computational identification of novel and conserved microRNAs in turmeric (Curcuma longa L.)

MicroRNAs (miRNAs) are endogenous, small, single stranded non‒coding 17‒24 nucleotide long RNAs that regulates gene expression in plants and animals either by direct degradation of mRNA or inhibition of translation. Conventional approaches employed for the detection of miRNA are costly and time consuming. On the other hand, comparative genomics assisted with modern tools of bioinformatics provide an efficient and cost‒effective identification of novel and conserved miRNAs via homology search within known miRNAs. The present study reports computational identification of miRNAs present in the Expressed Sequence Tags (ESTs) of turmeric (Curcuma longa L.), a plant known for its great medicinal and culinary value. A total of 12678 ESTs were assembled into 2710 contigs/unigenes and homology sequence search was performed against 4647 non‒redundant miRNAs of Viridiplantae. In total, 102 potential mature miRNAs showed homology with 51 contigs/unigenes. Subsequently, four novel miRNAs were identified in turmeric, which satisfied the potential miRNA criteria. These four miRNAs were further characterized for target gene prediction identifying 10 target genes with their putative functions. Our study provides a valuable resource on miRNAs in turmeric, and also suggests a methodology useful for the identification of novel and conserved miRNA in non‒model but important organisms, whose EST and genome sequence data are available in the public domain. Keywords: turmeric;, Curcuma longa, microRNA, expressed sequence tags

Genetics and mapping of the novel leaf-colour mutant gene

Leaf colour plays an important role in plant growth and development. In this study, a stably inherited yellow–green leaf mutation, temporarily designated yglw-1, was initially isolated from progenies of wheat (Triticum aestivum L.) cross Bainong58409/Caifumai. The yellow-leaf phenotype could be observed in the yglw-1 mutant during the seedling stage, and then the yellow leaves gradually progressed from the bottom to the top throughout development. Compared with its sister line Cf5019-21 with a normal leaf-colour phenotype, the main agronomic traits of the yglw-1 mutant including spike length, number of kernels per spike and 1000-kernel weight were significantly decreased. Measurement of chlorophyll contents showed that chlorophylls a and b in the third top leaf at the jointing stage and the penultimate leaf and third top leaf at the heading stage were obviously lower in yglw-1 than in Cf5019-21. Genetic analysis indicated that the yellow–green leaf trait was controlled by a single recessive gene. Bulked segregant analysis found that two published simple sequence repeat (SSR) markers and four expressed sequence tag-sequence tagged site (EST-STS) markers were linked to the yglw-1 gene, and BE494262 was the most closely linked marker to yglw-1, at a genetic distance of 2.3 cM. To increase the density of the genetic map, 135 novel SSR markers at a certain distance near the marker BE494262 were developed according to SSR loci detected in the Chinese Spring reference sequence. Of these, nine SSR markers detected polymorphic fragments between the two parents as well as between the contrasting DNA bulks. Finally, the yglw-1 gene was mapped between SSR markers Xhwm1 and Xhwm43 and showed genetic distances 1.9 cM and 23.9 cM on chromosome 2BS. These findings can facilitate candidate-gene prediction and map-based cloning of the yglw-1 gene.

Exploitation of Walnut (Juglans regia L.) Expressed Sequence Tags for Development of SSR Markers After In Silico Analysis

The walnut (Juglans regia) has been extensively characterized for expressed sequence tags (EST) sequences and currently 6169492 ESTs are available in National Center for Biotechnology Information. Although this is a valuable resource for marker development, the redundancy in sequences makes the mining out of unique candidates for designing markers cumbersome. Keeping this in view, the present study was undertaken with the aim to remove the data redundancy in walnut ESTs and then to develop simple sequence repeats (SSRs) markers. The EST sequences were assembled into a non-redundant set of 85 contigs and 1584 singletons (total sequences 1699), indicating 16.55% reduction in data redundancy. These 1699 sequences were then used to mine out SSR motifs. 132 EST-SSRs were detected, with dinucleotide repeats being predominant (70.45%), followed by trinucleotide repeats (27.27%) and very less frequent hexanucleotides (2.27%). These markers were validated by designing primer pairs. 15 of these designed primers were tested on a group of 37 walnut genotypes. Out of which 7 markers gave robust amplification, generating polymorphism. These findings indicate the usefulness of EST-SSRs in genome analysis. This study further emphasizes the importance of assembly of the vast amounts of data submitted in public databases. Our results have generated a set of non redundant walnut ESTs which is of prime importance for development of marker systems without any repetition or overlapping.