First, information about the quality of individual ESTs is available. Second, the sequences of multiple overlapping ESTs can be aligned. The alignment may help to pinpoint unwanted unmatching ESTs and sequencing errors, and may highlight intron sequences or unreported splice sites, which could guide future experimental work.
We thank Drs D. Lipman and G. Schuler for their insightful comments, J. Wootton for communicating results before publication, and D. Bassett,Jr, M. Boguski and G. Schuler for their critical reading of the manuscript. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account.
Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Materials and Methods. Results and Discussion. Wolfsberg , Tyra G. Oxford Academic. Google Scholar. Cite Cite Tyra G. Select Format Select format. Permissions Icon Permissions.
Open in new tab Download slide. Analysis of splicing of all matching ESTs from coupled and uncoupled clones The full length genomic sequences of these 15 human genes were retrieved using Entrez. Analysis of coupled and uncoupled clones based on types of matching and unmatching ESTs The full length genomic sequences of these 15 human genes were retrieved using Entrez.
Search ADS. Issue Section:. Download all slides. Comments 0. Add comment Close comment form modal. I agree to the terms and conditions. You must accept the terms and conditions. Add comment Cancel. Submit a comment. Comment title. You have entered an invalid code. Submit Cancel. Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion. Please check for further notifications by email. These results also were in accordance with those of previous studies.
In addition to these TF families, several others known to be involved in plant development were also present in our data. Leaf senescence is an integrated response of leaf cells to age and other internal and environmental signals. It is an exceptionally complex and dynamic genetic process [46]. Arabidopsis thaliana is a favorite model for the molecular genetic study of leaf senescence [47] — [49].
The LSD is also a platform to study leaf senescence [50]. During leaf senescence, nutrients in the leaf are reallocated to younger leaves, growing seeds, or other growing organs in a process of nutrient salvage, e. Many the genes involved in lipid metabolism function in leaf senescence.
Lipid-degrading enzymes, such as lytic acyl hydrolase, phosphatidic acid phosphatase, phospholipase D, and lipoxygenase appear to be involved in hydrolysis and metabolism of the membrane lipid in senescing leaves [51] , [52]. Changed expression of the Arabidopsis acyl hydrolase gene in transgenic plants led to altered leaf senescence phenotypes [53]. The hormonal pathways appear to affect all stages of leaf senescence.
In this work, numerous genes belonging to hormone response pathways were also identified. These results indicated that many previously-known leaf SAGs and pathways were included in this library. Three GhYLS genes were successfully cloned and analyzed. Their expression profiles revealed that their transcripts accumulated in leaves during senescence. Thus, these genes could potentially serve as molecular markers for distinguishing the complex regulatory networks of leaf senescence processes.
This library provides a robust sequence resource and will be a useful tool for cloning the full-length sequences of functional genes for further leaf senescence-related analysis in G. At the blooming stage, unexpanded leaves of the same size near the tops of stems were selected and marked. The day when leaves were fully expanded was considered the first day.
Leaves were collected every 5 d for 70 d. Then, clones were randomly selected and fully sequenced to test fullness ratios of the cDNA inserts of the library. Finally, qRT-PCR was used to estimate the relative concentration of a highly abundant clone in both the non-normalized and the normalized cDNA populations. Clones were randomly picked and transferred into well plates.
Sequences that passed the quality control screening for high-confidence base calls Q20 and with lengths longer than bp were defined as high quality EST and deposited into the dbESTs division of GenBank. To assign GO terms, functional annotation was performed using Blast2GO software based on sequence similarity [62] — [64]. To examine gene expressions during leaf development, the leaves used for qRT-PCR were harvested from approximately 10 individual plants for each stage.
Total chlorophyll of the samples was measured as described by Lichtenthaler [66]. Homologs of leaf senescence-related protein sequences were identified and randomly selected according to the LSD function annotation. The specific primer pairs for nine selected genes and the internal control gene actin are listed in Table S1. The identified clones were sequenced in both directions with the internal primers. The amino-acid multiple-sequence alignment was analyzed using GeneDoc.
Phylogenetic analysis was performed using the neighbor-joining method in MEGA 4 [68]. Performed the experiments: ML DL. Analyzed the data: ML DL. Wrote the paper: ML. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Background Cotton Gossypium hirsutum L. Introduction Cotton Gossypium spp. Download: PPT. Figure 1. Sequence length distribution of upland cotton ESTs after assembly. Figure 2. Frequency and distribution of ESTs among assembled contigs. Table 1. Summary statistics of EST data generated from 11, Gossypium hirsutum leaves. Table 2.
The most abundant ESTs detected in the Gossypium hirsutum leaf library. Table 3. Comparison of the Gossypium hirsutum leaf EST library with those of other species. Figure 3. Functional classifications of upland cotton 2, unigenes that were assigned with GO terms. Table 4. Table 5. Functional categories of Gossypium hirsutum leaf senescence-related genes a. Figure 4.
Expression patterns of nine putative leaf senescence related genes from upland cotton. Table 6. The most abundant putative transcriptional factors TFs. Figure 5. Phylogeny analysis of putative MYB transcription factors. Discussion Gossypium hirsutum is one of the most economically-important species in its genus. Supporting Information. Furthermore, two genotypes were used in this initial fl-cDNA library construction, one known to be drought tolerant BAT and the other which is the subject of full-length genomic sequencing G A total of nearly 10, ESTs were generated from the second library to show the utility of this technique in determining gene structure.
This EST sequencing project was performed as part of a breeding project to discover molecular markers in common beans for marginal areas of Sub-Saharan Africa and the process of marker discovery from full-length cDNA sequences is discussed. We also aimed to compare the ESTs from the full-length cDNA library to two previous large EST sets for common bean and show the advantages this technology has for genomic tool development in this less-well studied species.
Two elite common bean genotypes were selected based on their attributes for stress resistance and use in genomics studies. First, the Mesoamerican gene pool advanced lines BAT was selected based on its deep rooting ability and known drought tolerance [ 35 ], and second, the Andean gene pool genotype G was selected based on resistance to both Al-toxicity and low phosphorus soil stresses [ 36 , 37 ].
This latter genotype has been selected for whole genome sequencing based on the physical map made by Schlueter et al. The genotypes were subjected to drought and irrigated control conditions as main treatments. The experiment was established under greenhouse conditions using plastic PVC-tubes of 0.
The irrigation was stopped at 10 days after seed germination to simulate natural drought stress under all drought treatments. In a split plot design with 2 replicates, a control treatment was normally irrigated throughout the experiment for each soil type. Aerial and root tissues were harvested, washed and frozen immediately with liquid nitrogen for the subsequent total RNA isolations.
Harvests of tissues were performed at five-day intervals until reaching 35 days of drought period and sampling from each of the development stages of the plants: seedlings cotyledons and shoots , growing stage leaves, stems, shoots and roots , and reproductive stage flowers and small pods.
Roots were carefully obtained by washing away the sand-soil mixture with a light stream of water and then rinsing in a plastic tub. Tissues of the irrigated control were only collected at 15, 30, and 45 days after germination, which were representative of the stages of growing and flowering see Additional file 2 for explanation of the time course for tissue harvest and for a photograph of the deep root, cylinder culture system.
Frozen tissues were ground mechanically to a fine powder using liquid nitrogen. Aerial and root tissues with their corresponding treatments and sampling times were processed separately. After quantification each the amount of RNA obtained from each sampling time for the drought and irrigated treatments were pooled separately within each target genotype. Total RNA from aerial and root tissues were also pooled separately.
RNA quality was determined through denaturing agarose gels 1. Once transformed into Eschereschia coli bacteria, clones from the G library were picked by an automated robotic colony picker to a total of 10, clones half from each library. A total of individual clones were sized and sequenced from both ends at RIKEN to determine their insert size and quality before sending a total of 9, clones approximately half the library for sequencing at the Washington State University sequencing center in St.
Louis, Missouri. Although we made two libraries one Andean from G and one Mesoamerican from BAT we only sequenced from the first of these given funding constraints and given the relative importance of G which is being sequenced by a whole genome shotgun approach S.
Jackson, pers. Sequence reads were trimmed for low quality and vector contamination. These were generally found at the 3'end of the sequences and were discarded. Poly-A tails were identified and trimmed at their adjacent base if followed by vector sequences. Searches were made against corresponding non-redundant nr database with blastx against all higher plant proteins. The E-value and positive alignment length distributions were then determined with a high-scoring segment pair HSP cutoff of 33 and an E-value threshold of 1E Gene ontologies GO were assigned based on Harris et al.
Genes were then evaluated for their likely molecular cellular function, cellular localization and involvement at four gene ontology levels. In addition, KEGG annotation was also used to determine KO ontologies and to determine the position of the unigenes and singletons form the full assembly described above in various biochemical pathways and directed acyclic graphing DAG was used to determine the gene relationships.
Finally, simple sequence repeats were identified in the full-length sequences based on their locations within the 5'UTR or ORF using first the software program RepeatFinder [ 49 ] and then more definitively SciRoKO [ 33 ].
The libraries were based on totals of 3. For the libraries, a total of 20, clones were selected robotically half from each genotype and in preliminary sequencing of 5'and 3'ends the clones of both libraries a well plate each were shown to average 1. Additional file 3 shows the distribution of the initial clones in terms of total length. Following this initial testing, nearly 10, clones were sequenced from the Andean G library. The sequences were all from 5'ends with an average read length of nucleotides nt.
Of these, surpassed the initial threshold of low number of N's and had an average read length of nt. Upon vector trimming the average insert length was nt and the number of cleaned sequences after trimming and low quality sequence elimination was Sequences were submitted to GenBank entry numbers JKJK and consisted of both singletons and independent clones within each contig.
After assembly of all the full-length cDNA clones, a total of unigenes were identified. These consisted of singletons On average the number of sequences within a contig was 3. Meanwhile, the average length of the singletons was nt. The large number of singletons and the high number of contigs relative to the number of sequences indicated low redundancy for the library.
This was not surprising given that the plants for the RNA extraction had been grown in three different soil treatments and under both drought-stress and irrigated conditions and that whole plants above and below ground parts were harvested at seven and three timepoints for the cDNA preparations, respectively. This created scaffolds or full-length genes for the Thibivilliers et al. For the work of Thibivilliers et al. In the case of this analysis of re-assembly of Thibivilliers et al.
Homologies to genes from medicago unigenes, Example of alternate splicing and 5'end location of an EST for aquaporin from the full-length library.
The last arrow at the bottom of the figure shows the homology to the equivalent ortholog in the soybean genome. Top species hit for the full-length unigenes was with soybean over 1, unigenes and then grape over hits. These were followed by to hits each with medicago, poplar and castor bean. Only highest hits were directly with common bean genes. These numbers of hits reflect both the similarity of the species, especially in the case of common bean-soybean, and the number of unigenes that exist in GenBank under the non-redundant UniProtKB or TAIR databases which were most frequently used for mapping of unigene ontology Figure 4.
The most frequently expressed genes were generally housekeeping genes but did reflect the abiotic stress conditions for the plants from which the full-length library was made and sequenced Additional file 4 including the propensity of finding aquaporins which are useful for water uptake in cells under osmotic stress Figure 3.
Mapping databases with a top hits for gene ontology GO and b gene ontology evidence code distribution for blastx hits in terms of numbers of GO terms or sequences y-axes , respectively. Based on the full collection of unigenes from the full-length cDNA library sequencing project. In the gene ontology analysis Table 2 , various levels were evaluated and at level 2 for biological processes a quarter of the genes each fell into either cellular For molecular functions the genes were almost evenly divided between binding Cellular components were divided among cell, organelle and to a lesser extent cell membrane and extracellular regions.
These values were different in diversity compared to values for a recent set of root ESTs made in our laboratory [ 15 ]. For example response in the full-length library there were higher percentages of genes for biological regulation, developmental processes and response to stimuli.
Since the full-length library was made from aerial as well as below-ground tissue differences of this nature would be expected. Although important goals of any sequencing project may be to obtain a genomic sequence and identify a complete set of genes, the ultimate goal is to gain an understanding of when, where and how a gene is turned on, a process commonly referred to as gene expression.
Once we begin to understand where and how a gene is expressed under normal circumstances, we can then study what happens in an altered state, such as in disease. To accomplish the latter goal however, researchers must identify and study the protein, or proteins, coded for by a gene. As one can imagine, finding a gene that codes for a protein, or proteins, is not easy.
Traditionally, scientists would start their search by defining a biological problem and developing a strategy for researching the problem. Oftentimes, a search of the scientific literature provided various clues about how to proceed.
For example, other laboratories may have published data that established a link between a particular protein and a disease of interest. Researchers would then work to isolate that protein, determine its function, and locate the gene that coded for the protein. Alternatively, scientists could conduct what is referred to as linkage studies to determine the chromosomal location of a particular gene. Once the chromosomal location was determined, scientists would use biochemical methods to isolate the gene and its corresponding protein.
Either way, these methods took a great deal of time--years in some cases--and yielded the location and description of only a small percentage of the genes found in the human genome.
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