Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project
© Fernández et al; licensee BioMed Central Ltd. 2003
Received: 19 May 2003
Accepted: 30 September 2003
Published: 30 September 2003
Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits.
Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important agronomic traits and key regulatory and physiological genes.
The application of suppressed subtracted hybridization technology not only enabled the cost effective isolation of differentially expressed sequences but it also allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences when compared to major sequencing projects.
Cultivated sunflower (Helianthus annuus L.) is one of the most important sources of vegetable oil worldwide. During the last decade, rapid advances in applied genetics and genomic technologies have led to the development of saturated sunflower genetic maps based on different molecular markers including RFLP, AFLP and SSR [1–10]. More recently, large-scale cDNA sequencing projects have identified expressed sequence tags (ESTs) in different plant species. Today, more than 100 plant species are represented in the EST division (dbEST) of GenBank http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html with a total of 2,063,406 entries. However, Arabidopsis, rice, maize, tomato and soybean ESTs projects gather more than 50% of the total entries. The Compositae is represented by 113,149 entries, of which 44,961 correspond to sunflower ESTs. These projects allow the characterization of full sets of transcribed genes in the target organisms and provide, at the same time, a source of genetic markers that can be functionally associated to important agronomical traits reinforcing and complementing the use of anonymous markers. Thus, the use of EST-based markers could lead to genetic mapping of a gene that directly affects the trait or a specific sequence could be target due to its predicted function based on sequence comparison . ESTs generated from cDNA libraries should represent, ideally, all expressed genes in a target organ/tissue, at a specific developmental stage and/ or in a specific environment. However, differences in expression level among genes in a given tissue yield mRNAs that differ in abundance, making it difficult to capture rare mRNA in cDNA libraries. This problem also leads to redundant sequencing of clones representing the same expressed genes, affecting the efficiency and cost effectiveness of the EST approach  which hinders research laboratories with small budgets to perform EST characterization studies. To avoid this problem, different strategies based on normalized cDNA libraries have been reported in many different organisms [12–14] including plants [15–17]. In this study, we report for the first time in sunflower, the isolation and characterization of ESTs from organ-specific cDNA libraries constructed by suppressed subtractive hybridization  as an alternative to identify differentially expressed sunflower transcripts. We analyzed the efficiency of the subtraction and enrichment methods for each cDNA library generated and present the differential level of representation for functional EST groups based on Gene Ontology annotation , as well as a comprehensive description of individual non-redundant sequences generated.
Results and Discussion
Construction of organ-specific cDNA libraries
Number of isolated, analyzed, differential and non redundant sequences by organ-specific cDNA library.
R1 flower (1)
R4 Flower (2)
Differential sequences between (2) and (3) a,b
Non-redundant percentage b
Differential sequences c
Average ORF c
Average insert size (bp)b
Analysis of organ-specificity among non-redundant sequences confirmed that a high proportion of the non-redundant sequences in each library corresponded to sequences only detected in that tissue. In the R4 flower and stem cDNA libraries, 93.5% and 98.7% of the analyzed sequences were unique to those libraries, respectively (Table 1). A global analysis including all constructed libraries revealed that 87.8% of the generated non-redundant ESTs were indeed differentially expressed sequences.
EST analysis based on predicted gene function
A total of 190 sequences (60 %), out of 318 non-redundant ESTs, showed significant similarity to known gene sequences in the database with a stringency level (E value) of 10-3 and a score value higher than 80. No significant differences in average insert length in both were detected between the sequences that match previous entries on GenBank and those that did not show similarities. These results indicate that the lengths of the sequences reported in this study are good enough to retrieve significant hits in GenBank database. Out of the remaining 128 sequences (40 %) that exhibited no significant similarity to known genes, 90 sequences (70%) exhibited significant homology to ESTs with unknown function on public databases while the remaining sequences representing 32 % are new reported sequences. The Compositae is represented in the GenBank by 113,149 entries of which 44,961 correspond to recently deposit sunflower ESTs and the rest to lettuce (Lactuca sativa; Composite Genome Initiative, CGI, http://cgpdb.ucdavis.edu/database/cgpdb.php. Out of these 44,961 sunflower ESTs, 15,248 are unique sequences, and only circa 2,061 are functionally annotated sequences (TIGR Gene Indices, http://www.tigr.org/tdb/tgi/hagi. In spite of this extensive amount of available information, sequence comparison of the 318 non-redundant sequences generated in this study against 37,208 unique H. annuus and L. sativa sequences (HaGI and LsGI http://www.tigr.org/tdb/tgi/lsgi, TIGR) showed that 197 (59.9%) did not exhibit significant similarity to previously reported sunflower ESTs whilst 228 (69.3%) did not match L. sativa ESTs. The important level of homology found with other plant ESTs that do not belong to the Compositae family indicate that this fact was not due to highly variable or non-coding sequences present at the 3' end of the mRNA. Since the ESTs in this study are derived from polyA RNA and thus enriched in 3' end sequences of the mRNAs, while the ESTs recently deposited at the CGI are enriched in the 5' end of the mRNA, a comparative study of outcome BLASTX was performed in order to determine if the 197 newly detected sunflower genes were indeed represented at GenBank by previously deposited sunflower ESTs from different gene regions. This analysis revealed that most of these sequences share annotation but do not share identities at a nucleotide level, thus some of them are likely to be variants of gene families.
"Structural proteins" and "motor" as well as sequences related to cell growth and metabolisms, here included in the "enzyme" class, showed a low level of representation compared to the corresponding values obtained by non-normalized cDNA libraries [25, 26]. A similar under representation of ESTs from the cell metabolisms category was reported for other normalized cDNA libraries . This result shows that the normalization step that took place in the construction of the cDNA libraries was efficient in diminishing the level of highly abundant transcripts equally represented in the different analyzed tissues.
Differential ESTs related to response to biotic and/or abiotic stress
GO functional definitionb
Phosphoethanolamine N methytransferase
Putative phosphoethanolamine N methytrans
Fructose biphosphate aldolase
Cyclophilin-type peptidy-prolyl cis-trans isomerase
Cyclophilin (EC 126.96.36.199) (Peptidyl-prolyl cis-trans isomerase) (PPIase)
Electron transfer flavoprotein
Heat shock protein
Stromal 70 kDa heat shock-related protein
Fructose biphodphata aldolase
homologous to plastidic aldolases
Magnesium chelatase subunit
Magnesium chelatase subunit.
Oxygen-evolving enhancer protein 1
Proteasome subunit alpha type 2
Hydrolase, hydrolyzing O-glycosyl compounds
Germin-like protein 3
Polygalacturonase inhibitor protein
Immediate hypersensitivity response
Lipid transfer protein
Immediate hypersensitivity response
Lipid transfer protein
Putative cysteine proteinase
Peptidyl-prolyl cis-trans isomerase
Peptidylprolyl isomerase (Cyclophilin)
Putative zinc protease
Calcium ion binding
Response to biotic stimulus
Pathogenesis-related protein R major form
Spermidine synthase 1 (EC 188.8.131.52)
Cationic peroxidase 2
Chloroplast drought-induced stress protein of 32 kDa
Serine protease inhibitor
Protease inhibitor 2
Cysteine protease inhibitor
Chloroplast drought-induced stress protein of 32 kDa
Probable cysteine synthase B (CSASE B) protein (EC 184.108.40.206).
In the present study, the "binding" class is equally represented in all the analyzed libraries, although this class includes sequence with putative involment in diverse processes such as transcription and translation factors, ATPase and cation binding proteins. Within this group low abundant transcripts like those coding for transcription factors and homeotic factors were specially detected in the R4 flower cDNA library. ESTs with homology to genes coding for signalling enzymes as MAP protein kinase and serine/threonin phosphatase were only detected in the stem cDNA library (Figure 2b). The functional category of "transporter" is represented by sequences with similarity to carrier protein genes as ATP-binding cassette (ABC) and electron transporters that were mainly detected in the R4 flower library. ESTs with similarity to homeobox genes here included in "development" were only detected in the early flower bud cDNA library. The homeobox sequences isolated in this work did not show similarity to previously reported sunflower homeobox genes [37–39]. Preliminary results showed that some of the agronomical interesting sequences, including those putatively related to response to biotic and abiotic stress, revealed polymorphisms when used as genetic markers in the analysis of genetically segregant populations derived from the crossing of parental lines with contrasting biotic and abiotic stress resistance behaviour (not shown).
The application of suppressed subtracted hybridization technology for the detection of differential ESTs allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences in spite of the large number of ESTs that have been recently release. Particularly interesting was the detection of a significant number of ESTs related to response to both abiotic and biotic stresses, as well as low abundance transcripts with high similarity to homeobox genes, transcription factors and signalling component genes that were not represented in the sunflower EST division at the GenBank. The R4 flower cDNA library was the library that provided the largest number of novel genes in sunflower, whilst the R1 flower bud library was particularly enriched in defence related genes. The detection of these novel sequences could contribute to the development of EST-based markers for important agronomic traits such as resistance to pathogens and tolerance to different environmental stresses such as extreme temperatures and drought, which are aspects crucial for sunflower crop improvement in many of the cultivated areas in the world.
The application of suppressed subtracted hybridization technology enabled the isolation of a significant number of organ-specific sunflower ESTs and allowed the identification of novel sequences from a relative small number of analyzed sequences. Redundancy level and percent of novel sequence detection varied among differential libraries reinforcing the importance of a careful selection of both target and driver transcript population according to project aims. In this work the R4 flower cDNA library provided the largest number of novel genes in sunflower, whilst the R1 flower bud library was particularly enriched in defence related genes. Some of the novel sequences reputed here share annotation but do not share identities at a nucleotide level with sunflower ESTs on public databases and thus, they are likely to be variants of gene families. We report for the first time in sunflower a significant number of novel sequences related to responses to abiotic and biotic stresses as well as low abundant transcripts with high similarity to homeobox genes, transcription factors and signalling components.
Sunflower seedlings (public inbred line RHA89) were grown under controlled green house conditions (20–24°C and 16 h light/ 8 h dark cycle), and then transplanted to the field during the crop season to develop mature plants. Leaves, stems, and capitulum buds from 1 to 2 cm of diameter (early flower buds) and 3 to 4 cm of diameter (late flower buds) were harvested from two months old plants and immediately frozen in liquid nitrogen. Roots were harvested from 15 day old plants grown in sand under green house conditions. All samples were stored frozen at -80°C until processed.
Total and poly (A+) RNA isolation
Total RNA was extracted from approximately 2 g of tissue using TRIzol® reagent following manufacturer recommendations (InVitrogen, USA). Poly (A+) RNA was isolated from 200–500 μg of total RNA using NucleoTrap® System (Promega, USA). RNA integrity was analyzed by checking its electrophoretic mobility on 1.5 % agarose gels in ME buffer (400 mM MOPS, 100 mM Na acetate, 10 mM EDTA pH 8.0, in diethyl-pyrocarbonate treated water). mRNA quantification was performed by UV absorbance at 260 nm (GenQuant pro, Amersham-Pharmacia, UK).
Construction of cDNA libraries
Differential cDNA libraries were constructed from different tissues including leaves, stems, roots and flower buds and from different developmental stages (e.g. R1 and R4 according to the description of sunflower growth stages by Schneiter and Miller ) using PCR-Select cDNA Subtraction Kit® (Clontech, USA). Firstly, cDNA was synthesized from 0.5–2.0 μg of poly (A+) RNA from the two types of tissues being compared. The tester (target tissue) and driver (reference tissue) cDNAs were then digested with RsaI, that yields blunt end fragments of approximately 400 bp length in average. We defined different driver populations for the different specific libraries, depending on specific interests. Leaf cDNA collection was arrested against a late flower bud cDNA population. Stem early flower bud and root cDNA collections were arrested against a leaf cDNA population.
Both, tester and driver, cDNA populations were processed following manufacturer instructions, with some modifications. The tester cDNA was subdivided into two halves, and each half was ligated to different cDNA adaptors. Two hybridization rounds were performed with an excess of driver cDNA. Hybridization conditions were performed as recommended by the manufacturer. The resulted products were subjected to two cycles of PCR with adaptor targeted primers to amplify the desired differentially expressed sequences. Amplifications were performed on a PT-100 DNA thermocycler (MJ Research, USA). First PCR master mix contained 10x PCR reaction buffer, 0.2 mM dNTPs, 0.4 μM PCR primer 1 and Advantage cDNA polymerase (Clontech, USA). PCR was performed under the following conditions: 94°C (30 sec) denaturing step followed by 27 cycles each consisting of a denaturation step at 94°C (30 sec), an annealing step at 66°C (30 sec) and an elongation step at 72°C (10 min). The second PCR master mix contained 10x PCR reaction buffer, 0.2 mM dNTPs, 0.4 μM nested PCR primer 1, 0.4 μM nested PCR primer 2 and Advantage cDNA polymerase, PCR was run through 12 cycles each consisting of a denaturing step at 94°C (30 sec), an annealing step at 66° (30 sec) and an elongation step at 72°C (1.5 min).
cDNA molecules were size-selected and fractions larger than 250 bp were cloned non-directionally into the pGem-T-Easy Vector® (Promega, USA). Ligation was performed at 4°C for 48 h and the resulting ligation product was used to transform Escherichia coli (XL1-blue strain) by electroporation (Pulse Controller, BioRad, USA).
cDNA libraries were plated onto solid Luria Bertani (LB) medium containing ampicillin. Recombinant clones were selected by β-galactosidase activity in media containing X-GAL and IPTG. White colonies were randomly picked to 364 well plates containing Freezing Medium (36 mM K2HPO4, 13.2 mM KH2PO4, 1.7 mM sodium citrate, 0.4 mM MgSO4, 6.8 mM (NH4)2 SO4, in LB medium and 4.4 % glycerol), grown overnight and later stored at -70°C. Recombinant plasmids were isolated using REAL 96 prep kit (Qiagen, Germany) as recommended by the supplier. Insert sizes of individual recombinant clones were examined by electrophoresis of EcoRI digestion products on 1.2 % agarose gels in TAE buffer .
Sequencing and sequence analysis
Recombinant plasmids were single-pass sequenced from the T7 universal primer site at sequencing facilities (Laboratorio de Alta Complejidad, IMyZA – CICVyA – INTA Castelar, Argentina; Centro de Biologia Molecular e Engenharia Genética – CBMEG Universidade Estadual de Campinas, Sao Paulo, Brazil and/or Department of Plant Pathology, Kansas University). Reverse sequencing was performed from the SP6 primer site, only when the forward sequences failed or were uninformative due to a short length. The generated EST sequences were stored in a relational database in which both 5' and 3' sequences were equally represented. Vector and uninformative sequences were automatically removed using computer program routines. The processed sequence were output to FASTA formatted files and a pile up (Biopipeline®) step routine written by in-house staff (S.L., Bioaxioma S.A.) was applied to detect remaining vector artifacts by comparing against a full vector sequence database. Redundancy was also analyzed by means of a clustering systems running under an alpha version of Biopipeline®. This system displays a graphic matrix which aligns the top scoring hits sequences in a score matrix. Sequences that exhibited more than 80% identity over total large sequence were considered identical or closely related and were assigned to a specific group. Sequence alignment of those highly similar sequences was confirmed by sequence alignment programs (ClustalW ). Contig analysis of the grouped ESTs was done using the contig assembly program Cap3 .
Sequence similarities searches against different protein databases were conducted using Advanced BLAST program . Default BLAST parameter values were used except for the E value (E = 10-3). The top scoring hits were automatically annotated according to the putative function returned by BLASTX. Gene Ontology (GO) annotation was performed using the GOblet software package  and a GO term associated to each sequence showing a significant similarity hit by BLASTX against SWALL search was defined. Sequences comparison against plant division ESTs, HaGI and LsGI were performed locally using BLASTN. These datasets were downloaded from public databases and the "Standalone WWW BLAST Server" from the National Center for Biotechnology Information (NCBI; ftp://ftp.ncbi.nih.gov/blast).
We are grateful to Valeria Peralta for the greenhouse work, to Dr. Roberto Perazzo and Lic. Gustavo Guida for his assistance on Biopipeline® step routine development and to Lic. Alejandro D'Angelo for technical support on database programming routines. We thank Dr. Mariana del Vas for critical reading of the manuscript. This research was supported by the ANPCyT/FONCYT; BID 1201 AC/AR PID 024 and by ASAGIR, Argentina. Ing. Agr. P. Fernandez holds a doctoral fellowship from the University of Buenos Aires, Dr. R. Heinz and Dr. N. Paniego are career members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Dr. H.E. Hopp is a career member of the Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CIC) and Professor at the Facultad de Ciencias Exactas y Naturales, University of Buenos Aires (UBA).
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