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The first set of EST resource for gene discovery and marker development in pigeonpea (Cajanus cajan L.)
Raju, Nikku L
Gnanesh, Belaghihalli N
Lekha, Pazhamala
Jayashree, Balaji
Pande, Suresh
Hiremath, Pavana J
Byregowda, Munishamappa
Singh, Nagendra K
Varshney, Rajeev K
BMC Plant Biology
Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs).
A total of 16 cDNA libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to FW ('ICPL 20102' and 'ICP 2376') and SMD ('ICP 7035' and 'TTB 7') and a total of 9,888 (9,468 high quality) ESTs were generated and deposited in dbEST of GenBank under accession numbers GR463974 to GR473857 and GR958228 to GR958231. Clustering and assembly analyses of these ESTs resulted into 4,557 unique sequences (unigenes) including 697 contigs and 3,860 singletons. BLASTN analysis of 4,557 unigenes showed a significant identity with ESTs of different legumes (23.2-60.3%), rice (28.3%), Arabidopsis (33.7%) and poplar (35.4%). As expected, pigeonpea ESTs are more closely related to soybean (60.3%) and cowpea ESTs (43.6%) than other plant ESTs. Similarly, BLASTX similarity results showed that only 1,603 (35.1%) out of 4,557 total unigenes correspond to known proteins in the UniProt database (≤ 1E-08). Functional categorization of the annotated unigenes sequences showed that 153 (3.3%) genes were assigned to cellular component category, 132 (2.8%) to biological process, and 132 (2.8%) in molecular function. Further, 19 genes were identified differentially expressed between FW- responsive genotypes and 20 between SMD- responsive genotypes. Generated ESTs were compiled together with 908 ESTs available in public domain, at the time of analysis, and a set of 5,085 unigenes were defined that were used for identification of molecular markers in pigeonpea. For instance, 3,583 simple sequence repeat (SSR) motifs were identified in 1,365 unigenes and 383 primer pairs were designed. Assessment of a set of 84 primer pairs on 40 elite pigeonpea lines showed polymorphism with 15 (28.8%) markers with an average of four alleles per marker and an average polymorphic information content (PIC) value of 0.40. Similarly, in silico mining of 133 contigs with ≥ 5 sequences detected 102 single nucleotide polymorphisms (SNPs) in 37 contigs. As an example, a set of 10 contigs were used for confirming in silico predicted SNPs in a set of four genotypes using wet lab experiments. Occurrence of SNPs were confirmed for all the 6 contigs for which scorable and sequenceable amplicons were generated. PCR amplicons were not obtained in case of 4 contigs. Recognition sites for restriction enzymes were identified for 102 SNPs in 37 contigs that indicates possibility of assaying SNPs in 37 genes using cleaved amplified polymorphic sequences (CAPS) assay.
The pigeonpea EST dataset generated here provides a transcriptomic resource for gene discovery and development of functional markers associated with biotic stress resistance. Sequence analyses of this dataset have showed conservation of a considerable number of pigeonpea transcripts across legume and model plant species analysed as well as some putative pigeonpea specific genes. Validation of identified biotic stress responsive genes should provide candidate genes for allele mining as well as candidate markers for molecular breeding.
Related Articles
Gene discovery from Jatropha curcas by sequencing of ESTs from normalized and full-length enriched cDNA library from developing seeds
Natarajan, Purushothaman
Kanagasabapathy, Deepa
Gunadayalan, Gnanasekaran
Panchalingam, Jasintha
shree, Noopur
Sugantham, Priyanka Annabel
Singh, Kavita Kumari
Madasamy, Parani
BMC Genomics
Jatropha curcas L. is promoted as an important non-edible biodiesel crop worldwide. Jatropha oil, which is a triacylglycerol, can be directly blended with petro-diesel or transesterified with methanol and used as biodiesel. Genetic improvement in jatropha is needed to increase the seed yield, oil content, drought and pest resistance, and to modify oil composition so that it becomes a technically and economically preferred source for biodiesel production. However, genetic improvement efforts in jatropha could not take advantage of genetic engineering methods due to lack of cloned genes from this species. To overcome this hurdle, the current gene discovery project was initiated with an objective of isolating as many functional genes as possible from J. curcas by large scale sequencing of expressed sequence tags (ESTs).
A normalized and full-length enriched cDNA library was constructed from developing seeds of J. curcas. The cDNA library contained about 1 × 106 clones and average insert size of the clones was 2.1 kb. Totally 12,084 ESTs were sequenced to average high quality read length of 576 bp. Contig analysis revealed 2258 contigs and 4751 singletons. Contig size ranged from 2-23 and there were 7333 ESTs in the contigs. This resulted in 7009 unigenes which were annotated by BLASTX. It showed 3982 unigenes with significant similarity to known genes and 2836 unigenes with significant similarity to genes of unknown, hypothetical and putative proteins. The remaining 191 unigenes which did not show similarity with any genes in the public database may encode for unique genes. Functional classification revealed unigenes related to broad range of cellular, molecular and biological functions. Among the 7009 unigenes, 6233 unigenes were identified to be potential full-length genes.
The high quality normalized cDNA library was constructed from developing seeds of J. curcas for the first time and 7009 unigenes coding for diverse biological functions including oil biosynthesis were identified. These genes will serve as invaluable genetic resource for crop improvement in jatropha to make it an ideal and profitable crop for biodiesel production.
Related Articles
A comprehensive resource of drought- and salinity- responsive ESTs for gene discovery and marker development in chickpea (Cicer arietinum L.)
Varshney, Rajeev K
Hiremath, Pavana J
Lekha, Pazhamala
Kashiwagi, Junichi
Balaji, Jayashree
Deokar, Amit A
Vadez, Vincent
Xiao, Yongli
Srinivasan, Ramamurthy
Gaur, Pooran M
Siddique, Kadambot HM
Town, Christopher D
Hoisington, David A
BMC Genomics
Chickpea (Cicer arietinum L.), an important grain legume crop of the world is seriously challenged by terminal drought and salinity stresses. However, very limited number of molecular markers and candidate genes are available for undertaking molecular breeding in chickpea to tackle these stresses. This study reports generation and analysis of comprehensive resource of drought- and salinity-responsive expressed sequence tags (ESTs) and gene-based markers.
A total of 20,162 (18,435 high quality) drought- and salinity- responsive ESTs were generated from ten different root tissue cDNA libraries of chickpea. Sequence editing, clustering and assembly analysis resulted in 6,404 unigenes (1,590 contigs and 4,814 singletons). Functional annotation of unigenes based on BLASTX analysis showed that 46.3% (2,965) had significant similarity (≤1E-05) to sequences in the non-redundant UniProt database. BLASTN analysis of unique sequences with ESTs of four legume species (Medicago, Lotus, soybean and groundnut) and three model plant species (rice, Arabidopsis and poplar) provided insights on conserved genes across legumes as well as novel transcripts for chickpea. Of 2,965 (46.3%) significant unigenes, only 2,071 (32.3%) unigenes could be functionally categorised according to Gene Ontology (GO) descriptions. A total of 2,029 sequences containing 3,728 simple sequence repeats (SSRs) were identified and 177 new EST-SSR markers were developed. Experimental validation of a set of 77 SSR markers on 24 genotypes revealed 230 alleles with an average of 4.6 alleles per marker and average polymorphism information content (PIC) value of 0.43. Besides SSR markers, 21,405 high confidence single nucleotide polymorphisms (SNPs) in 742 contigs (with ≥ 5 ESTs) were also identified. Recognition sites for restriction enzymes were identified for 7,884 SNPs in 240 contigs. Hierarchical clustering of 105 selected contigs provided clues about stress- responsive candidate genes and their expression profile showed predominance in specific stress-challenged libraries.
Generated set of chickpea ESTs serves as a resource of high quality transcripts for gene discovery and development of functional markers associated with abiotic stress tolerance that will be helpful to facilitate chickpea breeding. Mapping of gene-based markers in chickpea will also add more anchoring points to align genomes of chickpea and other legume species.
Related Articles
Generation, Annotation and Analysis of First Large-Scale Expressed Sequence Tags from Developing Fiber of Gossypium barbadense L
Yuan, Daojun
Tu, Lili
Zhang, Xianlong
PLoS ONE
Cotton fiber is the world's leading natural fiber used in the manufacture of textiles. Gossypium is also the model plant in the study of polyploidization, evolution, cell elongation, cell wall development, and cellulose biosynthesis. G. barbadense L. is an ideal candidate for providing new genetic variations useful to improve fiber quality for its superior properties. However, little is known about fiber development mechanisms of G. barbadense and only a few molecular resources are available in GenBank.
Methodology and Principal Findings
In total, 10,979 high-quality expressed sequence tags (ESTs) were generated from a normalized fiber cDNA library of G. barbadense. The ESTs were clustered and assembled into 5852 unigenes, consisting of 1492 contigs and 4360 singletons. The blastx result showed 2165 unigenes with significant similarity to known genes and 2687 unigenes with significant similarity to genes of predicted proteins. Functional classification revealed that unigenes were abundant in the functions of binding, catalytic activity, and metabolic pathways of carbohydrate, amino acid, energy, and lipids. The function motif/domain-related cytoskeleton and redox homeostasis were enriched. Among the 5852 unigenes, 282 and 736 unigenes were identified as potential cell wall biosynthesis and transcription factors, respectively. Furthermore, the relationships among cotton species or between cotton and other model plant systems were analyzed. Some putative species-specific unigenes of G. barbadense were highlighted.
The ESTs generated in this study are from the first large-scale EST project for G. barbadense and significantly enhance the number of G. barbadense ESTs in public databases. This knowledge will contribute to cotton improvements by studying fiber development mechanisms of G. barbadense, establishing a breeding program using marker-assisted selection, and discovering candidate genes related to important agronomic traits of cotton through oligonucleotide array. Our work will also provide important resources for comparative genomics, polyploidization, and genome evolution among Gossypium species.
Related Articles
A new genomic resource dedicated to wood formation in Eucalyptus
Rengel, David
Clemente, Hélène San
Servant, Florence
Ladouce, Nathalie
Paux, Etienne
Wincker, Patrick
Couloux, Arnaud
Sivadon, Pierre
Grima-Pettenati, Jacqueline
BMC Plant Biology
Renowned for their fast growth, valuable wood properties and wide adaptability, Eucalyptus species are amongst the most planted hardwoods in the world, yet they are still at the early stages of domestication because conventional breeding is slow and costly. Thus, there is huge potential for marker-assisted breeding programs to improve traits such as wood properties. To this end, the sequencing, analysis and annotation of a large collection of expressed sequences tags (ESTs) from genes involved in wood formation in Eucalyptus would provide a valuable resource.
We report here the normalization and sequencing of a cDNA library from developing Eucalyptus secondary xylem, as well as the construction and sequencing of two subtractive libraries (juvenile versus mature wood and vice versa). A total of 9,222 high quality sequences were collected from about 10,000 cDNA clones. The EST assembly generated a set of 3,857 wood-related unigenes including 2,461 contigs (Cg) and 1,396 singletons (Sg) that we named 'EUCAWOOD'. About 65% of the EUCAWOOD sequences produced matches with poplar, grapevine, Arabidopsis and rice protein sequence databases. BlastX searches of the Uniref100 protein database allowed us to allocate gene ontology (GO) and protein family terms to the EUCAWOOD unigenes. This annotation of the EUCAWOOD set revealed key functional categories involved in xylogenesis. For instance, 422 sequences matched various gene families involved in biosynthesis and assembly of primary and secondary cell walls. Interestingly, 141 sequences were annotated as transcription factors, some of them being orthologs of regulators known to be involved in xylogenesis. The EUCAWOOD dataset was also mined for genomic simple sequence repeat markers, yielding a total of 639 putative microsatellites. Finally, a publicly accessible database was created, supporting multiple queries on the EUCAWOOD dataset.
In this work, we have identified a large set of wood-related Eucalyptus unigenes called EUCAWOOD, thus creating a valuable resource for functional genomics studies of wood formation and molecular breeding in this economically important genus. This set of publicly available annotated sequences will be instrumental for candidate gene approaches, custom array development and marker-assisted selection programs aimed at improving and modulating wood properties.
Related Articles
MELOGEN: an EST database for melon functional genomics
Gonzalez-Ibeas, Daniel
Blanca, José
Roig, Cristina
González-To, Mireia
Picó, Belén
Truniger, Verónica
Gómez, Pedro
Deleu, Wim
Caño-Delgado, Ana
Arús, Pere
Nuez, Fernando
Garcia-Mas, Jordi
Puigdomènech, Pere
Aranda, Miguel A
BMC Genomics
Melon (Cucumis melo L.) is one of the most important fleshy fruits for fresh consumption. Despite this, few genomic resources exist for this species. To facilitate the discovery of genes involved in essential traits, such as fruit development, fruit maturation and disease resistance, and to speed up the process of breeding new and better adapted melon varieties, we have produced a large collection of expressed sequence tags (ESTs) from eight normalized cDNA libraries from different tissues in different physiological conditions.
We determined over 30,000 ESTs that were clustered into 16,637 non-redundant sequences or unigenes, comprising 6,023 tentative consensus sequences (contigs) and 10,614 unclustered sequences (singletons). Many potential molecular markers were identified in the melon dataset: 1,052 potential simple sequence repeats (SSRs) and 356 single nucleotide polymorphisms (SNPs) were found. Sixty-nine percent of the melon unigenes showed a significant similarity with proteins in databases. Functional classification of the unigenes was carried out following the Gene Ontology scheme. In total, 9,402 unigenes were mapped to one or more ontology. Remarkably, the distributions of melon and Arabidopsis unigenes followed similar tendencies, suggesting that the melon dataset is representative of the whole melon transcriptome. Bioinformatic analyses primarily focused on potential precursors of melon micro RNAs (miRNAs) in the melon dataset, but many other genes potentially controlling disease resistance and fruit quality traits were also identified. Patterns of transcript accumulation were characterised by Real-Time-qPCR for 20 of these genes.
The collection of ESTs characterised here represents a substantial increase on the genetic information available for melon. A database (MELOGEN) which contains all EST sequences, contig images and several tools for analysis and data mining has been created. This set of sequences constitutes also the basis for an oligo-based microarray for melon that is being used in experiments to further analyse the melon transcriptome.
Related Articles
Analysis of 13000 unique Citrus clusters associated with fruit quality, production and salinity tolerance
Terol, Javier
Conesa, Ana
Colmenero, Jose M
Cercos, Manuel
Tadeo, Francisco
Agustí, Javier
Alós, Enriqueta
Andres, Fernando
Soler, Guillermo
Brumos, Javier
Iglesias, Domingo J
Götz, Stefan
Legaz, Francisco
Argout, Xavier
Courtois, Brigitte
Ollitrault, Patrick
Dossat, Carole
Wincker, Patrick
Morillon, Raphael
Talon, Manuel
BMC Genomics
Improvement of Citrus, the most economically important fruit crop in the world, is extremely slow and inherently costly because of the long-term nature of tree breeding and an unusual combination of reproductive characteristics. Aside from disease resistance, major commercial traits in Citrus are improved fruit quality, higher yield and tolerance to environmental stresses, especially salinity.
A normalized full length and 9 standard cDNA libraries were generated, representing particular treatments and tissues from selected varieties (Citrus clementina and C. sinensis) and rootstocks (C. reshni, and C. sinenis × Poncirus trifoliata) differing in fruit quality, resistance to abscission, and tolerance to salinity. The goal of this work was to provide a large expressed sequence tag (EST) collection enriched with transcripts related to these well appreciated agronomical traits. Towards this end, more than 54000 ESTs derived from these libraries were analyzed and annotated. Assembly of 52626 useful sequences generated 15664 putative transcription units distributed in 7120 contigs, and 8544 singletons. BLAST annotation produced significant hits for more than 80% of the hypothetical transcription units and suggested that 647 of these might be Citrus specific unigenes. The unigene set, composed of ~13000 putative different transcripts, including more than 5000 novel Citrus genes, was assigned with putative functions based on similarity, GO annotations and protein domains
Comparative genomics with Arabidopsis revealed the presence of putative conserved orthologs and single copy genes in Citrus and also the occurrence of both gene duplication events and increased number of genes for specific pathways. In addition, phylogenetic analysis performed on the ammonium transporter family and glycosyl transferase family 20 suggested the existence of Citrus paralogs. Analysis of the Citrus gene space showed that the most important metabolic pathways known to affect fruit quality were represented in the unigene set. Overall, the similarity analyses indicated that the sequences of the genes belonging to these varieties and rootstocks were essentially identical, suggesting that the differential behaviour of these species cannot be attributed to major sequence divergences. This Citrus EST assembly contributes both crucial information to discover genes of agronomical interest and tools for genetic and genomic analyses, such as the development of new markers and microarrays.
Related Articles
Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii
Weng, Jing-Ke
Tanurdzic, Milos
Chapple, Clint
BMC Genomics
The lycophyte Selaginella moellendorffii is a member of one of the oldest lineages of vascular plants on Earth. Fossil records show that the lycophyte clade arose 400 million years ago, 150–200 million years earlier than angiosperms, a group of plants that includes the well-studied flowering plant Arabidopsis thaliana. S. moellendorffii has a genome size of approximately 100 Mbp, as small or smaller than that of A. thaliana. S. moellendorffii has the potential to provide significant comparative information to better understand the evolution of vascular plants.
We sequenced 2181 Expressed Sequence Tags (ESTs) from a S. moellendorffii cDNA library. One thousand three hundred and one non-redundant sequences were assembled, containing 291 contigs and 1010 singletons. Approximately 75% of the ESTs matched proteins in the non-redundant protein database. Among 1301 clusters, 343 were categorized according to Gene Ontology (GO) hierarchy and were compared to the GO mapping of A. thaliana tentative consensus sequences. We compared S. moellendorffii ESTs to the A. thaliana and Physcomitrella patens EST databases, using the tBLASTX algorithm. Approximately 60% of the ESTs exhibited similarity with both A. thaliana and P. patens ESTs; whereas, 13% and 1% of the ESTs had exclusive similarity with A. thaliana and P. patens ESTs, respectively. A substantial proportion of the ESTs (26%) had no match with A. thaliana or P. patens ESTs.
We discovered 1301 putative unigenes in S. moellendorffii. These results give an initial insight into its transcriptome that will aid in the study of the S. moellendorffii genome in the near future.
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Massively parallel pyrosequencing-based transcriptome analyses of small brown planthopper (Laodelphax striatellus), a vector insect transmitting rice stripe virus (RSV)
Zhang, Fujie
Guo, Hongyan
Zheng, Huajun
Zhou, Tong
Zhou, Yijun
Wang, Shengyue
Fang, Rongxiang
Qian, Wei
Chen, Xiaoying
BMC Genomics
The small brown planthopper (Laodelphax striatellus) is an important agricultural pest that not only damages rice plants by sap-sucking, but also acts as a vector that transmits rice stripe virus (RSV), which can cause even more serious yield loss. Despite being a model organism for studying entomology, population biology, plant protection, molecular interactions among plants, viruses and insects, only a few genomic sequences are available for this species. To investigate its transcriptome and determine the differences between viruliferous and naïve L. striatellus, we employed 454-FLX high-throughput pyrosequencing to generate EST databases of this insect.
We obtained 201,281 and 218,681 high-quality reads from viruliferous and naïve L. striatellus, respectively, with an average read length as 230 bp. These reads were assembled into contigs and two EST databases were generated. When all reads were combined, 16,885 contigs and 24,607 singletons (a total of 41,492 unigenes) were obtained, which represents a transcriptome of the insect. BlastX search against the NCBI-NR database revealed that only 6,873 (16.6%) of these unigenes have significant matches. Comparison of the distribution of GO classification among viruliferous, naïve, and combined EST databases indicated that these libraries are broadly representative of the L. striatellus transcriptomes. Functionally diverse transcripts from RSV, endosymbiotic bacteria Wolbachia and yeast-like symbiotes were identified, which reflects the possible lifestyles of these microbial symbionts that live in the cells of the host insect. Comparative genomic analysis revealed that L. striatellus encodes similar innate immunity regulatory systems as other insects, such as RNA interference, JAK/STAT and partial Imd cascades, which might be involved in defense against viral infection. In addition, we determined the differences in gene expression between vector and naïve samples, which generated a list of candidate genes that are potentially involved in the symbiosis of L. striatellus and RSV.
To our knowledge, the present study is the first description of a genomic project for L. striatellus. The identification of transcripts from RSV, Wolbachia, yeast-like symbiotes and genes abundantly expressed in viruliferous insect, provided a starting-point for investigating the molecular basis of symbiosis among these organisms.
Related Articles
Isolation, identification and expression analysis of salt-induced genes in Suaeda maritima, a natural halophyte, using PCR-based suppression subtractive hybridization
Sahu, Binod B
Shaw, Birendra P
BMC Plant Biology
Despite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. Thus, there is a need of continued effort to understand the salt tolerance mechanism using suitable biotechnological techniques and test plants (species) to enable development of salt tolerant cultivars of interest. Therefore, the present study was undertaken to generate information on salt stress responsive genes in a natural halophyte, Suaeda maritima, using PCR-based suppression subtractive hybridization (PCR-SSH) technique.
Forward and reverse SSH cDNA libraries were constructed after exposing the young plants to 425 mM NaCl for 24 h. From the forward SSH cDNA library, 429 high quality ESTs were obtained. BLASTX search and TIGR assembler programme revealed overexpression of 167 unigenes comprising 89 singletons and 78 contigs with ESTs redundancy of 81.8%. Among the unigenes, 32.5% were found to be of special interest, indicating novel function of these genes with regard to salt tolerance. Literature search for the known unigenes revealed that only 17 of them were salt-inducible. A comparative analysis of the existing SSH cDNA libraries for NaCl stress in plants showed that only a few overexpressing unigenes were common in them. Moreover, the present study also showed increased expression of phosphoethanolamine N-methyltransferase gene, indicating the possible accumulation of a much studied osmoticum, glycinebetaine, in halophyte under salt stress. Functional categorization of the proteins as per the Munich database in general revealed that salt tolerance could be largely determined by the proteins involved in transcription, signal transduction, protein activity regulation and cell differentiation and organogenesis.
The study provided a clear indication of possible vital role of glycinebetaine in the salt tolerance process in S. maritima. However, the salt-induced expression of a large number of genes involved in a wide range of cellular functions was indicative of highly complex nature of the process as such. Most of the salt inducible genes, nonetheless, appeared to be species-specific. In light of the observations made, it is reasonable to emphasize that a comparative analysis of ESTs from SSH cDNA libraries generated systematically for a few halophytes with varying salt exposure time may clearly identify the key salt tolerance determinant genes to a minimum number, highly desirable for any genetic manipulation adventure.
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A full-length enriched cDNA library and expressed sequence tag analysis of the parasitic weed, Striga hermonthica
Yoshida, Satoko
Ishida, Juliane K
Kamal, Nasrein M
Ali, Abdelbagi M
Namba, Shigetou
Shirasu, Ken
BMC Plant Biology
The obligate parasitic plant witchweed (Striga hermonthica) infects major cereal crops such as sorghum, maize, and millet, and is the most devastating weed pest in Africa. An understanding of the nature of its parasitism would contribute to the development of more sophisticated management methods. However, the molecular and genomic resources currently available for the study of S. hermonthica are limited.
We constructed a full-length enriched cDNA library of S. hermonthica, sequenced 37,710 clones from the library, and obtained 67,814 expressed sequence tag (EST) sequences. The ESTs were assembled into 17,317 unigenes that included 10,319 contigs and 6,818 singletons. The S. hermonthica unigene dataset was subjected to a comparative analysis with other plant genomes or ESTs. Approximately 80% of the unigenes have homologs in other dicotyledonous plants including Arabidopsis, poplar, and grape. We found that 589 unigenes are conserved in the hemiparasitic Triphysaria species but not in other plant species. These are good candidates for genes specifically involved in plant parasitism. Furthermore, we found 1,445 putative simple sequence repeats (SSRs) in the S. hermonthica unigene dataset. We tested 64 pairs of PCR primers flanking the SSRs to develop genetic markers for the detection of polymorphisms. Most primer sets amplified polymorphicbands from individual plants collected at a single location, indicating high genetic diversity in S. hermonthica. We selected 10 primer pairs to analyze S. hermonthica harvested in the field from different host species and geographic locations. A clustering analysis suggests that genetic distances are not correlated with host specificity.
Our data provide the first extensive set of molecular resources for studying S. hermonthica, and include EST sequences, a comparative analysis with other plant genomes, and useful genetic markers. All the data are stored in a web-based database and freely available. These resources will be useful for genome annotation, gene discovery, functional analysis, molecular breeding, epidemiological studies, and studies of plant evolution.
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Characterization of an Atlantic cod (Gadus morhua) embryonic stem cell cDNA library
Olsvik, Pål A
Holen, Elisabeth
BMC Research Notes
The Atlantic cod is an ecologically and economically important North Atlantic fish species and also an emerging aquaculture species. To study gene expression in Atlantic cod embryonic stem (ES) cells, our goal was to generate and analyze expressed sequence tags (ESTs) from an ES cell cDNA library of mRNA consisting of approximately 3,900 ESTs.
We sequenced 3,935 EST clones using a directional cDNA library made from pooled ES cells harvested at the blastula stage. Quality filtering of these ESTs allowed identification of 2,719 high-quality sequences with an average length of 442 bp containing 368 contigs and 1,276 singletons (1,644 unique sequences). BLASTX searches produced 889 significant (E-value < 10-3) hits, of which 698 (42.5%) were annotated with Gene Ontology terms (E-value < 10-6). The number of unknown unique sequences was 946 (57.5%). All the high-quality EST sequences have been deposited in GenBank (GenBank: 2,719 sequences in UniGene library dbEST id: 22,021). Gene discovery and annotations are presented and discussed.
This set of ESTs represents one of the first attempts to describe mRNA in ES cells from a marine cold-water fish species, and provides a basis for gene expression studies of Atlantic cod ES cells.
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Comparative analysis of expressed sequence tags (ESTs) between drought-tolerant and -susceptible genotypes of chickpea under terminal drought stress
Deokar, Amit A
Kondawar, Vishwajith
Jain, Pradeep K
Karuppayil, S Mohan
Raju, N L
Vadez, Vincent
Varshney, Rajeev K
Srinivasan, R
BMC Plant Biology
Chickpea (Cicer arietinum L.) is an important grain-legume crop that is mainly grown in rainfed areas, where terminal drought is a major constraint to its productivity. We generated expressed sequence tags (ESTs) by suppression subtraction hybridization (SSH) to identify differentially expressed genes in drought-tolerant and -susceptible genotypes in chickpea.
EST libraries were generated by SSH from root and shoot tissues of IC4958 (drought tolerant) and ICC 1882 (drought resistant) exposed to terminal drought conditions by the dry down method. SSH libraries were also constructed by using 2 sets of bulks prepared from the RNA of root tissues from selected recombinant inbred lines (RILs) (10 each) for the extreme high and low root biomass phenotype. A total of 3062 unigenes (638 contigs and 2424 singletons), 51.4% of which were novel in chickpea, were derived by cluster assembly and sequence alignment of 5949 ESTs. Only 2185 (71%) unigenes showed significant BLASTX similarity (<1E-06) in the NCBI non-redundant (nr) database. Gene ontology functional classification terms (BLASTX results and GO term), were retrieved for 2006 (92.0%) sequences, and 656 sequences were further annotated with 812 Enzyme Commission (EC) codes and were mapped to 108 different KEGG pathways. In addition, expression status of 830 unigenes in response to terminal drought stress was evaluated using macro-array (dot blots). The expression of few selected genes was validated by northern blotting and quantitative real-time PCR assay.
Our study compares not only genes that are up- and down-regulated in a drought-tolerant genotype under terminal drought stress and a drought susceptible genotype but also between the bulks of the selected RILs exhibiting extreme phenotypes. More than 50% of the genes identified have been shown to be associated with drought stress in chickpea for the first time. This study not only serves as resource for marker discovery, but can provide a better insight into the selection of candidate genes (both up- and downregulated) associated with drought tolerance. These results can be used to identify suitable targets for manipulating the drought-tolerance trait in chickpea.
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Generation and analysis of expressed sequence tags from six developing xylem libraries in Pinus radiata D. Don
Li, Xinguo
Wu, Harry X
Dillon, Shannon K
Southerton, Simon G
BMC Genomics
Wood is a major renewable natural resource for the timber, fibre and bioenergy industry. Pinus radiata D. Don is the most important commercial plantation tree species in Australia and several other countries; however, genomic resources for this species are very limited in public databases. Our primary objective was to sequence a large number of expressed sequence tags (ESTs) from genes involved in wood formation in radiata pine.
Six developing xylem cDNA libraries were constructed from earlywood and latewood tissues sampled at juvenile (7 yrs), transition (11 yrs) and mature (30 yrs) ages, respectively. These xylem tissues represent six typical development stages in a rotation period of radiata pine. A total of 6,389 high quality ESTs were collected from 5,952 cDNA clones. Assembly of 5,952 ESTs from 5' end sequences generated 3,304 unigenes including 952 contigs and 2,352 singletons. About 97.0% of the 5,952 ESTs and 96.1% of the unigenes have matches in the UniProt and TIGR databases. Of the 3,174 unigenes with matches, 42.9% were not assigned GO (Gene Ontology) terms and their functions are unknown or unclassified. More than half (52.1%) of the 5,952 ESTs have matches in the Pfam database and represent 772 known protein families. About 18.0% of the 5,952 ESTs matched cell wall related genes in the MAIZEWALL database, representing all 18 categories, 91 of all 174 families and possibly 557 genes. Fifteen cell wall-related genes are ranked in the 30 most abundant genes, including CesA, tubulin, AGP, SAMS, actin, laccase, CCoAMT, MetE, phytocyanin, pectate lyase, cellulase, SuSy, expansin, chitinase and UDP-glucose dehydrogenase. Based on the PlantTFDB database 41 of the 64 transcription factor families in the poplar genome were identified as being involved in radiata pine wood formation. Comparative analysis of GO term abundance revealed a distinct transcriptome in juvenile earlywood formation compared to other stages of wood development.
The first large scale genomic resource in radiata pine was generated from six developing xylem cDNA libraries. Cell wall-related genes and transcription factors were identified. Juvenile earlywood has a distinct transcriptome, which is likely to contribute to the undesirable properties of juvenile wood in radiata pine. The publicly available resource of radiata pine will also be valuable for gene function studies and comparative genomics in forest trees.
Related Articles
Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)
Blair, Matthew W
Fernandez, Andrea C
Ishitani, Manabu
Moreta, Danilo
Seki, Motoaki
Ayling, Sarah
Shinozaki, Kazuo
BMC Plant Biology
Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean.
Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes.
The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean.
Related Articles
Analysis of the goldfish Carassius auratus olfactory epithelium transcriptome reveals the presence of numerous non-olfactory GPCR and putative receptors for progestin pheromones
Kolmakov, Nikolay N
Kube, Michael
Reinhardt, Richard
Canario, Adelino VM
BMC Genomics
The goldfish (Carassius auratus) uses steroids and prostaglandins as pheromone cues at different stages of the reproductive cycle to facilitate spawning synchronization. Steroid progestin pheromone binding has been detected in goldfish olfactory membranes but the receptors responsible for this specific binding remain unknown. In order to shed some light on the olfactory epithelium transcriptome and search for possible receptor candidates a large set of EST from this tissue were analysed and compared to and combined with a similar zebrafish (Danio rerio) resource.
We generated 4,797 high quality sequences from a normalized cDNA library of the goldfish olfactory epithelium, which were clustered in 3,879 unique sequences, grouped in 668 contigs and 3,211 singletons. BLASTX searches produced 3,243 significant (E-value < e-10) hits and Gene Ontology (GO) analysis annotated a further 1,223 of these genes (37.7%). Comparative analysis with zebrafish olfactory epithelium ESTs revealed 1,088 identical unigenes. The transcriptome size of both species was estimated at about 16,400 unigenes, based on the proportion of genes identified involved in Glucose Metabolic Process. Of 124 G-protein coupled receptors identified in the olfactory epithelium of both species, 56 were olfactory receptors. Beta and gamma membrane progestin receptors were also isolated by subcloning of RT-PCR products from both species and an olfactory epithelium specific splice form identified.
The high similarity between the goldfish and zebrafish olfactory systems allowed the creation of a 'cyprinid' olfactory epithelium library estimated to represent circa 70% of the transcriptome. These results are an important resource for the identification of components of signalling pathways involved in olfaction as well as putative targets for pharmacological and histochemical studies. The possible function of the receptors identified in the olfactory system is described. Moreover, the role of olfactory epithelium specific isoforms of classical membrane progestin receptor genes as candidates for preovulatory pheromone sensing is discussed.
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An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish
Reading, Benjamin J
Chapman, Robert W
Schaff, Jennifer E
Scholl, Elizabeth H
Opperman, Charles H
Sullivan, Craig V
BMC Research Notes
The striped bass and its relatives (genus Morone) are important fisheries and aquaculture species native to estuaries and rivers of the Atlantic coast and Gulf of Mexico in North America. To open avenues of gene expression research on reproduction and breeding of striped bass, we generated a collection of expressed sequence tags (ESTs) from a complementary DNA (cDNA) library representative of their ovarian transcriptome.
Sequences of a total of 230,151 ESTs (51,259,448 bp) were acquired by Roche 454 pyrosequencing of cDNA pooled from ovarian tissues obtained at all stages of oocyte growth, at ovulation (eggs), and during preovulatory atresia. Quality filtering of ESTs allowed assembly of 11,208 high-quality contigs ≥ 100 bp, including 2,984 contigs 500 bp or longer (average length 895 bp). Blastx comparisons revealed 5,482 gene orthologues (E-value < 10-3), of which 4,120 (36.7% of total contigs) were annotated with Gene Ontology terms (E-value < 10-6). There were 5,726 remaining unknown unique sequences (51.1% of total contigs). All of the high-quality EST sequences are available in the National Center for Biotechnology Information (NCBI) Short Read Archive (GenBank: SRX007394). Informative contigs were considered to be abundant if they were assembled from groups of ESTs comprising ≥ 0.15% of the total short read sequences (≥ 345 reads/contig). Approximately 52.5% of these abundant contigs were predicted to have predominant ovary expression through digital differential display in silico comparisons to zebrafish (Danio rerio) UniGene orthologues. Over 1,300 Gene Ontology terms from Biological Process classes of Reproduction, Reproductive process, and Developmental process were assigned to this collection of annotated contigs.
This first large reference sequence database available for the ecologically and economically important temperate basses (genus Morone) provides a foundation for gene expression studies in these species. The predicted predominance of ovary gene expression and assignment of directly relevant Gene Ontology classes suggests a powerful utility of this dataset for analysis of ovarian gene expression related to fundamental questions of oogenesis. Additionally, a high definition Agilent 60-mer oligo ovary 'UniClone' microarray with 8 × 15,000 probe format has been designed based on this striped bass transcriptome (eArray Group: Striper Group, Design ID: 029004).
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Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds
Shi, Cheng-Ying
Yang, Hua
Wei, Chao-Ling
Yu, Oliver
Zhang, Zheng-Zhu
Jiang, Chang-Jun
Sun, Jun
Li, Ye-Yun
Chen, Qi
Xia, Tao
Wan, Xiao-Chun
BMC Genomics
Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes.
Using high-throughput Illumina RNA-seq, the transcriptome from poly (A)+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR).
An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.
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Analysis of expressed sequence tags from Prunus mume flower and fruit and development of simple sequence repeat markers
Li, Xiaoying
Shangguan, Lingfei
Song, Changnian
Wang, Chen
Gao, Zhihong
Yu, Huaping
Fang, Jinggui
BMC Genetics
Expressed Sequence Tag (EST) has been a cost-effective tool in molecular biology and represents an abundant valuable resource for genome annotation, gene expression, and comparative genomics in plants.
In this study, we constructed a cDNA library of Prunus mume flower and fruit, sequenced 10,123 clones of the library, and obtained 8,656 expressed sequence tag (EST) sequences with high quality. The ESTs were assembled into 4,473 unigenes composed of 1,492 contigs and 2,981 singletons and that have been deposited in NCBI (accession IDs: GW868575 - GW873047), among which 1,294 unique ESTs were with known or putative functions. Furthermore, we found 1,233 putative simple sequence repeats (SSRs) in the P. mume unigene dataset. We randomly tested 42 pairs of PCR primers flanking potential SSRs, and 14 pairs were identified as true-to-type SSR loci and could amplify polymorphic bands from 20 individual plants of P. mume. We further used the 14 EST-SSR primer pairs to test the transferability on peach and plum. The result showed that nearly 89% of the primer pairs produced target PCR bands in the two species. A high level of marker polymorphism was observed in the plum species (65%) and low in the peach (46%), and the clustering analysis of the three species indicated that these SSR markers were useful in the evaluation of genetic relationships and diversity between and within the Prunus species.
We have constructed the first cDNA library of P. mume flower and fruit, and our data provide sets of molecular biology resources for P. mume and other Prunus species. These resources will be useful for further study such as genome annotation, new gene discovery, gene functional analysis, molecular breeding, evolution and comparative genomics between Prunus species.
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Generation and analysis of 9792 EST sequences from cold acclimated oat, Avena sativa
Bräutigam, Marcus
Lindlöf, Angelica
Zakhrabekova, Shakhira
Gharti-Chhetri, Gokarna
Olsson, Björn
Olsson, Olof
BMC Plant Biology
Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required.
From an oat cDNA library 9792 single-pass EST sequences were obtained. The library was prepared from pooled RNA samples isolated from leaves of four-week old Avena sativa (oat) plants incubated at +4°C for 4, 8, 16 and 32 hours. Exclusion of sequences shorter than 100 bp resulted in 8508 high-quality ESTs with a mean length of 710.7 bp. Clustering and assembly identified a set of 2800 different transcripts denoted the Avena sativa cold induced UniGene set (AsCIUniGene set). Taking advantage of various tools and databases, putative functions were assigned to 1620 (58%) of these genes. Of the remaining 1180 unclassified sequences, 427 appeared to be oat-specific since they lacked any significant sequence similarity (Blast E values > 10-10) to any sequence available in the public databases. Of the 2800 UniGene sequences, 398 displayed significant homology (BlastX E values ≤ 10-10) to genes previously reported to be involved in cold stress related processes. 107 novel oat transcription factors were also identified, out of which 51 were similar to genes previously shown to be cold induced. The CBF transcription factors have a major role in regulating cold acclimation. Four oat CBF sequences were found, belonging to the monocot cluster of DREB family ERF/AP2 domain proteins. Finally in the total EST sequence data (5.3 Mbp) approximately 400 potential SSRs were found, a frequency similar to what has previously been identified in Arabidopsis ESTs.
The AsCIUniGene set will now be used to fabricate an oat biochip, to perform various expression studies with different oat cultivars incubated at varying temperatures, to generate molecular markers and provide tools for various genetic transformation experiments in oat. This will lead to a better understanding of the cellular biology of this important crop and will open up new ways to improve its agronomical properties.
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EST analysis in Ginkgo biloba: an assessment of conserved developmental regulators and gymnosperm specific genes
Brenner, Eric D
Katari, Manpreet S
Stevenson, Dennis W
Rudd, Stephen A
Douglas, Andrew W
Moss, Walter N
Twigg, Richard W
Runko, Suzan J
Stellari, Giulia M
McCombie, WR
Coruzzi, Gloria M
BMC Genomics
Ginkgo biloba L. is the only surviving member of one of the oldest living seed plant groups with medicinal, spiritual and horticultural importance worldwide. As an evolutionary relic, it displays many characters found in the early, extinct seed plants and extant cycads. To establish a molecular base to understand the evolution of seeds and pollen, we created a cDNA library and EST dataset from the reproductive structures of male (microsporangiate), female (megasporangiate), and vegetative organs (leaves) of Ginkgo biloba.
RNA from newly emerged male and female reproductive organs and immature leaves was used to create three distinct cDNA libraries from which 6,434 ESTs were generated. These 6,434 ESTs from Ginkgo biloba were clustered into 3,830 unigenes. A comparison of our Ginkgo unigene set against the fully annotated genomes of rice and Arabidopsis, and all available ESTs in Genbank revealed that 256 Ginkgo unigenes match only genes among the gymnosperms and non-seed plants – many with multiple matches to genes in non-angiosperm plants. Conversely, another group of unigenes in Gingko had highly significant homology to transcription factors in angiosperms involved in development, including MADS box genes as well as post-transcriptional regulators. Several of the conserved developmental genes found in Ginkgo had top BLAST homology to cycad genes. We also note here the presence of ESTs in G. biloba similar to genes that to date have only been found in gymnosperms and an additional 22 Ginkgo genes common only to genes from cycads.
Our analysis of an EST dataset from G. biloba revealed genes potentially unique to gymnosperms. Many of these genes showed homology to fully sequenced clones from our cycad EST dataset found in common only with gymnosperms. Other Ginkgo ESTs are similar to developmental regulators in higher plants. This work sets the stage for future studies on Ginkgo to better understand seed and pollen evolution, and to resolve the ambiguous phylogenetic relationship of G. biloba among the gymnosperms.
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Transcriptome analysis of the sex pheromone gland of the noctuid moth Heliothis virescens
Vogel, Heiko
Heidel, Andrew J
Heckel, David G
Groot, Astrid T
BMC Genomics
The chemical components of sex pheromones have been determined for more than a thousand moth species, but so far only a handful of genes encoding enzymes responsible for the biosynthesis of these compounds have been identified. For understanding the evolution of moth sexual communication, it is essential to know which genes are involved in the production of specific pheromone components and what controls the variation in their relative frequencies in the pheromone blend. We used a transcriptomic approach to characterize the pheromone gland of the Noctuid moth Heliothis virescens, an important agricultural pest, in order to obtain substantial general sequence information and to identify a range of candidate genes involved in the pheromone biosynthetic pathway.
To facilitate identifying sets of genes involved in a broad range of processes and to capture rare transcripts, we developed our majority of ESTs from a normalized cDNA library of Heliothis virescens pheromone glands (PG). Combining these with a non-normalized library yielded a total of 17,233 ESTs, which assembled into 2,082 contigs and 6,228 singletons. Using BLAST searches of the NR and Swissprot databases we were able to identify a large number of putative unique gene elements (unigenes), which we compared to those derived from previous transcriptomic surveys of the larval stage of Heliothis virescens. The distribution of unigenes among GO Biological Process functional groups shows an overall similarity between PG and larval transcriptomes, but with distinct enrichment of specific pathways in the PG. In addition, we identified a large number of candidate genes in the pheromone biosynthetic pathways.
These data constitute one of the first large-scale EST-projects for Noctuidae, a much-needed resource for exploring these pest species. Our analysis shows a surprisingly complex transcriptome and we identified a large number of potential pheromone biosynthetic pathway and immune-related genes that can be applied to population and systematic studies of Heliothis virescens and other Noctuidae.
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Oil palm (Elaeis guineensis Jacq.) tissue culture ESTs: Identifying genes associated with callogenesis and embryogenesis
Low, Eng-Ti L
Alias, Halimah
Boon, Soo-Heong
Shariff, Elyana M
Tan, Chi-Yee A
Ooi, Leslie CL
Cheah, Suan-Choo
Raha, Abdul-Rahim
Wan, Kiew-Lian
Singh, Rajinder
BMC Plant Biology
Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes.
A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs). The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames.
This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However, in order to confirm whether these genes are suitable as early markers for embryogenesis, the genes need to be tested on earlier stages of tissue culture and a wider range of genotypes. This collection of ESTs is an important resource for genetic and genome analyses of the oil palm, particularly during tissue culture development.
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Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticus infection
Guo, Baozhu
Chen, Xiaoping
Dang, Phat
Scully, Brian T
Liang, Xuanqiang
Holbrook, C Corley
Yu, Jiujiang
Culbreath, Albert K
BMC Developmental Biology
Peanut (Arachis hypogaea L.) is an important crop economically and nutritionally, and is one of the most susceptible host crops to colonization of Aspergillus parasiticus and subsequent aflatoxin contamination. Knowledge from molecular genetic studies could help to devise strategies in alleviating this problem; however, few peanut DNA sequences are available in the public database. In order to understand the molecular basis of host resistance to aflatoxin contamination, a large-scale project was conducted to generate expressed sequence tags (ESTs) from developing seeds to identify resistance-related genes involved in defense response against Aspergillus infection and subsequent aflatoxin contamination.
We constructed six different cDNA libraries derived from developing peanut seeds at three reproduction stages (R5, R6 and R7) from a resistant and a susceptible cultivated peanut genotypes, 'Tifrunner' (susceptible to Aspergillus infection with higher aflatoxin contamination and resistant to TSWV) and 'GT-C20' (resistant to Aspergillus with reduced aflatoxin contamination and susceptible to TSWV). The developing peanut seed tissues were challenged by A. parasiticus and drought stress in the field. A total of 24,192 randomly selected cDNA clones from six libraries were sequenced. After removing vector sequences and quality trimming, 21,777 high-quality EST sequences were generated. Sequence clustering and assembling resulted in 8,689 unique EST sequences with 1,741 tentative consensus EST sequences (TCs) and 6,948 singleton ESTs. Functional classification was performed according to MIPS functional catalogue criteria. The unique EST sequences were divided into twenty-two categories. A similarity search against the non-redundant protein database available from NCBI indicated that 84.78% of total ESTs showed significant similarity to known proteins, of which 165 genes had been previously reported in peanuts. There were differences in overall expression patterns in different libraries and genotypes. A number of sequences were expressed throughout all of the libraries, representing constitutive expressed sequences. In order to identify resistance-related genes with significantly differential expression, a statistical analysis to estimate the relative abundance (R) was used to compare the relative abundance of each gene transcripts in each cDNA library. Thirty six and forty seven unique EST sequences with threshold of R > 4 from libraries of 'GT-C20' and 'Tifrunner', respectively, were selected for examination of temporal gene expression patterns according to EST frequencies. Nine and eight resistance-related genes with significant up-regulation were obtained in 'GT-C20' and 'Tifrunner' libraries, respectively. Among them, three genes were common in both genotypes. Furthermore, a comparison of our EST sequences with other plant sequences in the TIGR Gene Indices libraries showed that the percentage of peanut EST matched to Arabidopsis thaliana, maize (Zea mays), Medicago truncatula, rapeseed (Brassica napus), rice (Oryza sativa), soybean (Glycine max) and wheat (Triticum aestivum) ESTs ranged from 33.84% to 79.46% with the sequence identity ≥ 80%. These results revealed that peanut ESTs are more closely related to legume species than to cereal crops, and more homologous to dicot than to monocot plant species.
The developed ESTs can be used to discover novel sequences or genes, to identify resistance-related genes and to detect the differences among alleles or markers between these resistant and susceptible peanut genotypes. Additionally, this large collection of cultivated peanut EST sequences will make it possible to construct microarrays for gene expression studies and for further characterization of host resistance mechanisms. It will be a valuable genomic resource for the peanut community. The 21,777 ESTs have been deposited to the NCBI GenBank database with accession numbers ES702769 to ES724546.
Related Articles
Generation and analysis of expressed sequence tags from NaCl-treated Glycine soja
Ji, Wei
Li, Yong
Li, Jie
Dai, Cui-hong
Wang, Xi
Bai, Xi
Cai, Hua
Yang, Liang
Zhu, Yan-ming
BMC Plant Biology
Salinization causes negative effects on plant productivity and poses an increasingly serious threat to the sustainability of agriculture. Wild soybean (Glycine soja) can survive in highly saline conditions, therefore provides an ideal candidate plant system for salt tolerance gene mining.
As a first step towards the characterization of genes that contribute to combating salinity stress, we constructed a full-length cDNA library of Glycine soja (50109) leaf treated with 150 mM NaCl, using the SMART technology. Random expressed sequence tag (EST) sequencing of 2,219 clones produced 2,003 cleaned ESTs for gene expression analysis. The average read length of cleaned ESTs was 454 bp, with an average GC content of 40%. These ESTs were assembled using the PHRAP program to generate 375 contigs and 696 singlets. The resulting unigenes were categorized according to the Gene Ontology (GO) hierarchy. The potential roles of gene products associated with stress related ESTs were discussed. We compared the EST sequences of Glycine soja to that of Glycine max by using the blastn algorithm. Most expressed sequences from wild soybean exhibited similarity with soybean. All our EST data are available on the Internet (GenBank_Accn: DT082443~DT084445).
The Glycine soja ESTs will be used to mine salt tolerance gene, whose full-length cDNAs will be obtained easily from the full-length cDNA library. Comparison of Glycine soja ESTs with those of Glycine max revealed the potential to investigate the wild soybean's expression profile using the soybean's gene chip. This will provide opportunities to understand the genetic mechanisms underlying stress response of plants.
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Definition of Connolly, Cyril in English:
Connolly, Cyril
(1903–74), English writer and journalist; full name Cyril Vernon Connolly. His works include one novel, The Rock Pool (1936), and collections of essays, aphorisms, and reflections, among which are Enemies of Promise (1938) and The Unquiet Grave (1944).
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Penny gold mining stocks can return tremendous profits as the price of gold moves higher. Bonanza Goldfields, BONZ, is one stock to invest in here.
The company's new C.E.O. was a director at the U.S. Mint. Gold production is expected to begin soon at the company's Tarantula site.
Google "Dr. Madan Singh".
Read the recent Yahoo news.
"Penny stock today. Dollar stock tomorrow."


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Posted: 5/31/2010Provided by Citysearch -
My boyfriend and I went to Vines to celebrate our 3 year anniversary. From the moment we got to the valet guy all the way untill we left, the experience was nothing less than spectacular. The valet service was quick and personable. The hostesses were beaming with a smile on their face, showing their pride in their job. We were seated right away to a quiet corner of the dining room where we enjoyed wine recommendations by our servers. We ended up picking a sparkling wine and ordered steaks. The food was absolutely tasty and mouth watering! Steaks were cooked just right with the right amount of flavor! Needless to say the service and food is great and my boyfriend and I have found our new favorite anniversary spot! Thanks Vines!


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- Anônimo
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The owners, Shirley and Collin, really made this a great place to stay. They had a nice breakfast for us everyday and were very personable. Shirley even went so far as to help me with my laundry!
Aplicativos de Celular Hostelworld
Faça a reserva no caminho com os novos aplicativos móveis da Hostelworld.


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A better idea of what came inside the leave behinds I made.
All made by hand. (Minus the business cards printed by Mama's Sauce.)
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I have just bought a 15 inch macbook pro and need to connect it to my old 13 inch white macbook (2007 model), which cable should I use ?
Cable query on connection and migratio of data between macbooks old and new
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I used a firewire cable when I did it, I hooked it up and when you start the old macbook you hold down the T key while you power it up. This tells it you want to transfer all your files. There will be prompts on the screen, just like any other mac operation, it walks you through. Easy as pie.
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Last update: Wednesday 5 August 2015 [371 news items]
Large-size LCD shipments, revenues increase 11% in 2Q11, says DisplaySearch
Aug 18, 16:54
Shipments of large-area TFT LCD panels increased to 180.8 million units in second quarter 2011, growing by 11% on-quarter and 6% on-year. Revenues reached US$19.3 billion, also with...
Shipments of mobile PCs in China forecast to outpace desktops in 2013, says DisplaySearch
Jul 27, 16:55
According to the latest research from the DisplaySearch Quarterly China PC Shipment and Forecast Report, total China PC market shipments, which include desktops, all-in-one (AIO)...
Lower energy costs over the lifetime of the TV will pay back LED premium, says DisplaySearch
Jul 19, 14:44
Brands are missing an opportunity to market LED-backlit LCD TVs based on cost savings. According to DisplaySearch, analysis of electricity and TV prices together with power consumption...
Touch panel revenues to soar 90% in 2011, says DisplaySearch
Jul 13, 15:48
Total touch screen module revenues will reach US$13.4 billion in 2011, and nearly double in six years, reaching US$23.9 billion by 2017, according to DisplaySearch.
Connected TV shipments to exceed 138 million units in 2015, says DisplaySearch
Jul 6, 14:31
Products launched by TV manufacturers in 2011 show how critical internet services are to the future of TV. In 2011, more than 25% of all flat panel TVs shipped are expected to have...
LCD TV shipment growth rate drops to 9% in 1Q11, says DisplaySearch
Jun 7, 15:47
Global shipments of LCD TVs were 44.3 million units in the first quarter of 2011, an increase of 9% from the same period in 2010, according to DisplaySearch. On-year growth slowed...
3D TV panel shipments soar 104% in 1Q11, says DisplaySearch
Jun 1, 13:43
DisplaySearch anticipates that shipments of 3D LCD TV panels will account for 16.8% of total LCD TV panels shipped in the fourth quarter of 2011, and 12.3% for all of the year.
Panel prices for 2H May slightly up for monitors, TVs, says DisplaySearch
May 23, 10:20
Quotes for TFT-LCD panels used in monitors for the second half of May 2011 have increased US$1, while those used in TVs have risen by US$1-3, according to DisplaySearch.
Large-size TFT-LCD shipments decline 7%, revenues fall 12% in 1Q11, says DisplaySearch
May 12, 10:29
First-quarter 2011 shipments of large-area TFT LCD (larger than 9.1-inch) fell to 164 million units, down 7% on quarter, but up 5% on year. Revenues for the quarter were US$17.5 billion,...
LG display takes the lead in 1Q11 LED-backlit TV panel shipments; Samsung leads in revenues, says DisplaySearch
May 11, 15:50
Global shipments of LCD TV panels with LED backlights reached 16.4 million units in the first quarter of 2011, a slight decrease from 16.6 million units in the fourth quarter of 2010,...
DisplaySearch: Global LCD TV shipment growth rate to drop to 13% in 2011
May 6, 01:30
Global shipments of LCD TVs are expected to top 217 million units in 2011, representing a moderate growth of 13% from the levels recorded in 2010, in which global LCD TV shipments...
Mobile phone display revenues to reach US$22.4 billion in 2012, says DisplaySearch
Apr 20, 10:26
According DisplaySearch, revenues for mobile displays are forecast to grow 47% in 2011 and 19% in 2012, reaching US$22.4 billion in 2012.
LG, TCL, Hisense, Skyworth and Vizio to show double-digit LCD TV production growth from 1Q11 to 2Q11, says DisplaySearch
Apr 8, 10:45
Although the LCD TV supply chain is going through an inventory adjustment, major LCD TV brands are planning to increase production in second-quarter 2011 in order to catch up with...
Large-size LCD panel prices to remain flat in 1H April, says DisplaySearch
Apr 8, 01:10
Quotes for large-size LCD panels will remain flat in the first half of April, with 19-inch widescreen panels maintaining at US$60 on average and 21.5-inch widescreen models at US$75,...
Sony takes top position for flat panel TV shipments in India for 2010, says DisplaySearch
Mar 8, 15:54
Sony overtook Samsung Electronics and LG Electronics for the top position, with 22.1% of flat panel TVs shipped in the Indian market in 2010, according to DisplaySearch. The total...
LED TV panel shipments reach 16.6 million units in 4Q10, says DisplaySearch
Mar 1, 16:51
Global shipments of LCD TV panels with LED backlights reached a high of 16.6 million units in the fourth quarter of 2010, with strong growth in edge-lit configurations, according...
Handset display shipments up 6% to 413 million units in 3Q10, says DisplaySearch
Feb 25, 11:28
Handset display shipments increased 6% sequentially to 413.2 million units in the third quarter of 2010, while its revenues grew even faster at 13% sequentially to US$3.38 billion...
Global TV shipment growth improves to 15% on year in 4Q10 as LCD share surges, says DisplaySearch
Feb 23, 11:33
Global TV shipment growth improved in the fourth quarter of 2010 after falling sharply in the third quarter of 2010, rising 15% on year to a record 77.6 million units, according to...
Late February panel quotes remain flat for notebook, monitor use, slightly fall for TV use, says DisplaySearch
Feb 22, 01:40
International quotes for TFT-LCD panels for late February overall remain unchanged from previous quotes for use in LCD monitors and notebook PCs, but drop by US$1-3 for LCD TV panels,...
Large-size TFT-LCD shipments grow 26% on year in 2010 to 665 million units, says DisplaySearch
Feb 15, 15:44
Global large-size TFT-LCD panel shipments reached a record high of 665 million units in 2010, with strong growth in TV, notebook, tablet PC, and public display applications, according...
Manufacturing equipment spending for LTPS-based FPDs to explode in 2011, says DisplaySearch
Feb 9, 11:33
Spending on equipment used to make low temperature polysilicon (LTPS)-based displays is expected to surge to an all-time high of US$2.4 billion in 2011, according to DisplaySearch.
Plasma TV panel shipments hit record high in 4Q10, says DisplaySearch
Feb 8, 16:17
Plasma TV panel shipments increased 1% sequentially in the fourth quarter of 2010 and 9% on year to 5.2 million units, according to DisplaySearch. Major plasma makers operated at...
Tablet PCs to grow 200% to 55.7 million units in 2011, says DisplaySearch
Feb 8, 10:14
Momentum behind tablet (slate) PC shipments is growing, with an on-year increase of nearly 200% to 55.7 million units expected in 2011, according to DisplaySearch. Though growth rates...
Large-size LCD panel pices to continue to fall for TVs, with concerns for another slight drop for IT devices in January, says DisplaySearch
Jan 26, 14:17
Weak demand in the end-product markets seems to be slowing price movements of large-size LCD panels. In December, notebook panel prices remained unchanged or rose by 1% from the previous...
Shipments of cell LCD TV panels to increase 30% in 2011, says DisplaySearch
Jan 25, 15:42
In an effort to meet growing customer demand, LCD panel manufacturers are planning to grow their cell businesses 30% in 2011 to support the increasing needs of their TV manufacturer...
Inventory of LCD panel makers returned to healthy levels at the end of 2010, says DisplaySearch
Jan 21, 15:04
The level of inventory in panel makers' fabs and in the sales pipeline is a key variable for TFT LCD supply and demand, and panel makers' inventories returned to a healthy level at...
LCD glass substrates demand to go up 11%, capacity 13% in 2011, says DisplaySearch
Jan 19, 11:36
As the transition to flat panel TV nears completion, TFT-LCD TV panel demand is slowing, from 41% on year in 2010 to 8.3% in 2011. Total TFT-LCD panel area demand is expected to increase...
Global LCD TV market grows 31% in 2010, slowing to 13% in 2011, says DisplaySearch
Jan 4, 15:12
Global TV shipments in 2010 will reach more than 247 million units, an increase of 17% from 2009 and the best growth seen since the start of the flat-panel TV transition, according...
Connected TVs forecast to account for 21% of global TV shipments in 2010, says DisplaySearch
Dec 31, 11:53
While the consumer electronics industry prepares its wares for the CES 2011 in Las Vegas, the foundations of a quiet revolution in TV viewing continue to be built, with 21% of all...
Global touch panel market to grow more than 30% on year in 2010, says DisplaySearch
Dec 30, 15:01
The global touch panel industry is on track for more than 30% on year growth in 2010, according to DisplaySearch.
LCD TV panel manufacturers developing unique specifications for emerging markets, says DisplaySearch
Dec 23, 15:39
LCD TV panel manufacturers are introducing advanced features and enhancements such as 3D, 480Hz frame rates, ultra-slim form factors, and direct-lit LED backlights, but they are also...
LED TV panel prices see significant drop, monitor and notebook panel prices remain flat in 2H December, says DisplaySearch
Dec 22, 17:08
Prices for LCD TV panels continue to drop in the second half of December with more significant price drop for LED-backlit models, while prices for monitor and notebook panels remain...
Korea panel utilization rate reaches over 90% in November, says DisplaySearch
Dec 14, 14:09
Korea-based TFT-LCD panel makers' average utilization rates reached over 90% in November, while Taiwan-based panel makers were gradually increasing their utilization rates mainly...
LG Electronics captures top share of flat panel TV shipments in India, says DisplaySearch
Dec 10, 16:04
LG Electronics increased its unit share of flat panel TV shipments to 22.7% in the third quarter of 2010, taking the top market share position in India's TV market, Samsung Electronics...
Most important fab for LCD TV panel production is 8G, says DisplaySearch
Dec 6, 14:30
As panel makers expand, they are increasingly utilizing 8G fabs for LCD TV panel production, and the production of LCD TV panels at 8G increased to 41% in the third quarter of 2010,...
Macronix remains in the red
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Diabetes Health Center
Hugh Herr
"From that experience," he says, "because of Albert Dow, I felt compelled to make the best of it."
At the beginning, his work dealt primarily with elderly patients, most of whom had lost limbs to diabetes. Now, though, Herr's work is partially funded through the Veterans Administration.
"At Walter Reed you see all these young guys who are in top physical shape and who are very motivated," he says. "We're looking at how we can build limbs that are more robust, that don't break as easily, and that allow faster locomotion." Some of Herr's patients already have brought his prostheses back to the war zone with them.
"There's an odd dialogue in this world," he muses. "You say things like, 'Well, I forgot my foot,' and it doesn't compute for some people." Recently Herr developed a prosthesis that suits him.
It is a long, slender rod connected to something that looks very much like a tiny stock-car engine atop a flat artificial foot. With a small smile, Hugh Herr walks across the quiet laboratory over to where his new ankle is.
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Tree teachers
Two Henrico residents played key roles with Dominion's Project Plant It! program this year. Molly Nugent, a third-grade student at Rivers Edge Elementary, served as the student spokesperson and appeared on several television and radio shows to share what she learned about trees and the environment. Andy Hunt (Godwin Class of 2004) played the role of Ranger Spruce Sassafras for Radio Disney's Tree Trivia assemblies held at 20 schools throughout Virginia. During the assemblies, students cheered for trees while answering trivia questions in a game-show format hosted by the Ranger.
For more information about Project Plant It!, an environmental education program that aligns with State Standards of Learning for math, science and other subjects, visit http://www.projectplantit.com.
Local choral group seeks volunteers
Citizen Staff Reports 07/18/2016
Good Vibrations choral group is looking for all voices with some musical experience to sing with the volunteer choir. The season runs from August to November. Genre includes patriotic and inspirational. > Read more.
County plans Fourth of July event
Henrico County Recreation and Parks will present "Red, White, and Lights" at Meadow Farm Museum/Crump Park July 4.
Henrico County has hosted a Fourth of July celebration annually since 1981, but this year's event will offer a later start time and expanded hours and be highlighted by new entertainment.
The free event will begin at 4:30 p.m. and will feature the Richmond Symphony, a laser-light show, patriotic performances, and family activities. > Read more.
Restaurant Watch
Find out how your favorite dining establishments fared during their most recent inspections by the Virginia Department of Health. > Read more.
Henrico CASA seeks volunteers for training as child advocates
'Evening with Henry Winkler' event to benefit Henrico Schools
Henrico Public Library offers new nature exploration backpacks
Henrico Extension to hold free lawn care workshop Aug. 13
Allen named COO of Parham Doctors' Hospital
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Jul. 21, 2016Click here to read the print edition.
Restaurant Watch
Weekend Top 10
Good food and fun, bargain prices
Weekend Top 10
Polka taking over Henrico libraries July 19-21
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Obituary – Angela Carole Shumate
Obituary – Samuel A. "Sammy" Kirby
Obituary – Virginia Whipple Seaton
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Local choral group seeks volunteers
County plans Fourth of July event
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'Good old days' were good – for some
Of character and parenting
Opinion: Rename school for late Henrico officer
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Henrico teams win district, state championships
Varina HS to host Basketball Skill Development Academy June 20-23
Strawberry visits Tuckahoe Little League
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CalendarThe Henrico County Community Author Showcase, a program that connects writers and readers in the community, will begin at 7 p.m. and continue on Thursdays at various libraries. Paula Harrison… Full text


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Hippie tattoos reflect the era of the 1960's.
This body art captures the characteristics and look of hippies; the layouts are attractive and are expressions of peace and love.
One can say without a doubt that floral tattoos make awesome hippie tats and daisy tattoos are very popular with the girls.


text:
In a powerful dissent this morning, Justice Sotomayor had some extremely critical words for her colleagues
In her dissent of the Supreme Court's decision today to dismiss a case concerning the death of Israel Leija, Jr., Justice Sonia Sotomayor criticized her colleagues for sanctioning a "shoot first, think later approach to policing."
Leija was shot to death in 2010 during a high-speed police chase on I-27 in Texas.According to NBC News, the chase began after a local officer tried to arrest Leija on an outstanding warrant at a fast food restaurant. During the 18-minute-long chase, Leija twice called police dispatch to warn he was armed and willing to shoot if the chase wasn't called off.
Police laid spike strips at three spots along the highway in an attempt to disable Leija's car. Despite orders from a superior to stand by until Leija reached the spike strips, State Trooper Chadrin Mullenix fired 6 shots from an overpass at the speeding car. At least 4 of these shots fatally hit Leija; none hit the car's engine block.
"[I]t was clearly established under the Fourth Amendment," Sotomayor wrote in her dissent, "that an officer in Mullenix's position should not have fired the shots."
"Mullenix took his shot when Leija was between 25 and 30 yards away from the spike strip, traveling at 85 miles per hour," Sotomayor explained. "Even if his shots hit Leija's engine block, the car would not have stopped instantly."
After the shooting Mullenix said to his superior officer, "How's that for proactive?" And though "the comment does not impact our legal analysis," Sotomayor said, "[T]he comment seems to me revealing of the culture this court's decision supports when it calls it reasonable — or even reasonably reasonable — to use deadly force for no discernible gain and over a supervisor's express order to 'stand by.'"
The Court's decision could play a substantial role in shaping the way police misconduct cases are tried in the future.
Find the full RawStory report here.


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Sweet & Spicy Cucumbers
Total Time
Prep 20 mins
Cook 0 mins
After a shopping trip to Costco I had an over abundance of English cucumbers and red bell peppers so I came up with this recipe. I love things that are sweet, tangy, and spicy so it seemed like the natural thing to make. This has become a favorite of mine and a big hit among friends & family!
Ingredients Nutrition
- 2 English cucumbers, sliced very thin
- 1 table spoon salt
- 1⁄4 onion, sliced very thin
- 1⁄4 fresh red bell pepper, sliced very thin (don't use green bell pepper)
- 1⁄4 cup mirin
- 1⁄8 cup soy sauce
- 1⁄2 cup rice wine vinegar
- 2 tablespoons sugar (honey, Splenda, or jam will also work)
- 1 teaspoon sesame oil
- 1 teaspoon mongolian hot oil (or just add a pinch of crushed red pepper flakes)
- 1⁄4 teaspoon cayenne pepper
- 1⁄4 teaspoon black pepper
- 1 -2 tablespoon sesame seeds
- In a large bowl combine sliced cucumber + 2 tablespoons of salt and set aside.
- Slice onion and bell pepper and place in a separate bowl. To this bowl add the ingredients for the dressing and stir to combine.
- Rinse the cucumber under cold water and drain.
- Combine cucumbers, onion, pepper and dressing and stir to coat veggies then store in an air-tight container in the refrigerator for up to 2 weeks.
Most Helpful
These cucumbers are very good and a bit spicy. I only made half the dressing, but probably needed just 1/4 of what the recipes calls for. We didn't want this to be too sweet, so I added only 1/2 teaspoon of sugar, which was just right for us.
If only I could rate this recipe with more stars!! Its a 10 all the way! I had this recipe EXACTLY as written, and I suggest DO NOT mess with perfection! Try it at least once without alterations and I promise you won't be disappointed. Sweet, spicy, crunchy, devine goodness! Thanks Merry Cook for the recipe! Its a new family favorite!


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Last edited by jeffy; 10-03-2011 at 02:01 PM. Reason: 404
Just got dumped at least for tonigt from Monday night football. Not sure if he was censored by ESPN or the NFL for stating his personal opinion.
Don't say anything bad about "THE CHOSE ONE" or else! Big brother is watching!
Hihope Hiland Heathen of Perth CD, RE, CGC, TDI. (RIP)
teracroft Clan Piper of Perth CGC TDI (RIP)
Boyne's Fast Break of Perth CGC ( munchin in training)
Dropping Hank Jr for good had to be a move by ABC/Disney/ESPN with the NFL's blessing.
Had they not, tonight's game would have been cancelled for lack of players.
"I don't believe there should be any restrictions when it comes to firearms. None."
"""A couple of pleasant but obscure former governors representing a minor party may sound like an unpromising use of your ballot. Until you consider the alternatives." Gary Johnson
M&K's HR UH Tucker of Texoma JH
M&K's SHR Prime Black Angus
M&K's Miss Jessie Girl JH
Sir Jacob of Lakeview-Jake
Freeway JYD
Mike Whitworth


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Message Subject
Walmat FREEZING ALL AMMO...IN ALL STORES!!!
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A conversation overheard by a friend of mine in a Wal Mart store. My friend observed two boxes of ammo on the shelves and was wondering why so little. Another man came in and stood near him and called for the manager. The man said, "I have been buying my ammo here for years. Why is there nothing left on the shelves?" The manager said, "I don't know, we get in new shipments all the time and someone buys them up." The man said, "buys them up?" "How many boxes do they buy at one time?" The manager said, "hundreds at a time." The man said, "who is, why so many at once, is it several people?" The manager said, "look, all I know is we restock our shelves with the last shipment at midnight and someone comes in and buys hundreds of boxes." "I don't know if it is more than one person."
Being a conspiracy theorist, it just makes me wonder who is it that is cleaning the shelves of ammo out at night when everyone is asleep? Do they have an agenda?
maybe some rich guy is putting his stock portfolio into bullets, seems like a good investment to me.
I hope there on our side.
What you say is the correct answer, I am just wondering if it is the right answer. Yes, people want ammo, but maybe some "group" doesn't want them to have ammo?
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