Isolation and sequence analysis of oak and spruce cDNA clones

Introduction

The adaptation of individuals as well as populations to the environment is not well understood because of the lack of knowledge of the genes involved in these processes.  The highly developed and so far preferentially used marker systems, such as RAPDs, AFLPs, nuclear SSRs, and rDNA ITS, mainly represent repetitive regions of the nuclear genome, with the rDNA markers being further confined to the nucleolar organizer regions (NOR).  These neutral markers are ideal for assessing the genetic diversity as well as evolutionary relationship of populations, since the distribution of their variation is presumably not influenced by selective forces.  Close linkage of any important genes controlling adaptive characters with molecular markers currently available can only be a fortuitous and rare occurrence.  There is therefore a need to develop additional DNA polymorphisms representing genes involved in adaptive processes; such non-neutral markers could indicate the differentiation of populations on the basis of selective and adaptive features.  The recently developed DNA microarray technology could facilitate the discovery of genes modulated by environmental changes (Schena et al., 1995), thus identifying genes involved in adaptation processes.  However, this technology requires the availability of cDNA (complementary DNA) sequences to be tested for differential expression under various environmental conditions.  Complementary DNA sequences are the representatives of the messenger RNA (mRNA) transcripts of the expressed genes.  It is also known that useful sequence variation for polymorphic DNA markers can often be found at non-translated parts of mRNA sequences (Gil et al. 1997). Therefore, here we describe the isolation and sequence characterisation of cDNA clones randomly selected from cDNA libraries of oak and spruce.

Material and Methods

Tissue culture

Suspension cultures of a locally established Quercus petraea embryonic line were maintained in P24 medium.  For osmotic treatment the medium was diluted four times with distilled water.  Preliminary experiments showed that cell growth was not significantly influenced under such nutrition conditions for several weeks (not shown).

Isolation of total RNA from plant cells

The modified method of Cathala et al. (1983) was used as follows: 9.2g oak tissue culture was homogenized in liquid N₂ until it became fine powder.  30ml of lysis buffer (5M guanidium monothiocyanate dissolved at 60°C, 10M EDTA, 50 mM Tris pH 7.5, 8% ß-Mercaptoethanol) were added to the frozen samples and carefully mixed during melting (the sample was divided into aliquots to be able to handle the volume).  Then the samples were centrifuged at 10,000 rpm. for 20 min. Afterwards the supernatant was transferred into sterile 34ml Beckman centrifuge tubes, four volumes of 7M LiCl were added and incubated at 4°C overnight (or for a minimum of 12 hours).  The samples were centrifuged 90 min. at 14,000 rpm at 4°C in a Sorvall S34 rotor.  The pellets were resuspended in 5ml 3M LiCl and centrifuged again for 60 min. at 15,000 rpm. 3ml solubilisation buffer (0.1% SDS, 1mM EDTA, 10mM Tris pH7.5) were added to dissolve the pellets.  The samples were frozen again and vortexed during melting.  Then the RNA was extracted with an equal volume of phenol and then phenol-chloroform. Subsequently 0.1 volume of 3M NaAc (pH 4.9) was added and the chloroform extraction was repeated.  The samples were precipitated with 2.5 volumes of ethanol (abs.) at -70°C for 2 hours, then centrifuged for 20 min. at 10,000 rpm and washed with 80% Ethanol (chilled to -20°).  The pellets were dried under vacuum and dissolved in a total volume of 400 µl solubilisation buffer.  The RNA concentration was determined by photometer at 230 and 260nm wavelengths, and finally 2µg was checked on a 1.5% sterile agarose gel in 1x TBE.

Poly A+ RNA purification

The poly A+ RNA was purified by Dynabeads (Dynabeads Oligo(dT)25) according to the manufacturer's specifications.

Library Construction:

The oak cDNA libraries were constructed with Clontech's SMART® PCR cDNA Library Construction Kit according to the instructions of the user manual.  The parameters of the libraries are presented in Table 1.

The Norway spruce cDNA library originates from photomixotrophic suspension cells of Picea abies (L.) Karst.  PolyA+ RNA was isolated after treatment with a fungal elicitor (Galliano et al., 1993).

DNA sequencing

The selected clones were sequenced on an ABI 373XL sequencer, using the ABI Prism's BigDye® Terminator Cycle Sequencing Ready Reaction Kit with the following modification: Instead of 8µl Terminator Ready Reaction Mix only 4µl plus 4µl halfBigDye (Sigma) were taken, and 4 instead of 3.2 pmol of each primer were used.  Nearly all identified clones were sequenced to full length on both DNA strands by an oligonucleotide walking strategy.

BLAST search

The obtained oak DNA sequences were compared directly to DNA sequence databases by the BLAST 2.0 search system (NCBI).  They were also translated to putative amino acid sequences and then compared to the Swissprot database. The Norway spruce nucleotide sequences were compared to all main public databases using the network WU-BLAST similarity search server of the Swiss Institute for Experimental Cancer Research.

Results and discussion

Oak

One untreated and two osmotic shock induced cDNA libraries have been established from Quercus petraea tissue culture cells.  The initial clone number of the libraries was about 3 x 106 each (Table 1) which allows the isolation of cDNA clones representing low copy number mRNA species as well.  The average length of inserts in the cDNA clones varied from 792 to 881 basepairs.  In the present study fifty randomly selected clones were picked from each library and their insert size established. Clones containing inserts shorter than 500 bp were discarded.  The remaining 82 clones were sequenced mostly to full length.  Sixty out of the 82 clones proved to contain an appropriate 3' end of the mRNA represented by the presence of the poly A tail (Table 2).  The analysed clones have an average insert size of 0.9 kb.  The putative identity of 27 clones (45%) could be established by the BLAST sequence comparison system. As far as protein function is concerned, it was possible to identify six ribosomal protein sequences (three 60S /clones 6, 31, 70/ and 40S /clones 21, 75, 76/ each) possibly representing abundant mRNA species.  Two heat shock (clones 43, 168) and two lipid transfer proteins could also be identified (clones 82, 92).  The rest of the putative proteins represent single proteins of a different function (Table 2).

Norway spruce

One-hundred-thirty cDNA clones were sequenced from the fungal elicitor-induced spruce cDNA library.  Based on significant homologies with known genes of other organisms, 30% of the spruce clones were identified as housekeeping genes and putative stress-related genes, encoding a broad spectrum of metabolic pathways.  Using the sequence data, PCR primer pairs were designed in order to amplify expressed sequence tag (EST) sites in Picea abies.  For the 18 trees tested, all primer pairs yielded PCR bands matching the size exactly predicted from our cDNA data.  In the case of 11 PCR primer pairs, polymorphic amplification patterns were seen in diploid bud DNA extracts. Seven EST markers detected co-dominant inheritance by comparing the banding pattern obtained from the diploid bud-DNA extract to the corresponding haploid megagametophytes.  The remaining markers revealed polymorphic bands in the megagametophyte samples, confirming the existence of multigene families. Our results indicate that the number of alleles, which were identified at each locus within a population of 100 trees, varies between two and five.  Such markers are suitable tools for the verification of genetic variation within populations and corresponding forest reproductive material.  Furthermore, such markers may be utilised in the monitoring of viability selection and genetic loads such as inbreeding.

Conclusions

We could establish three cDNA libraries of oak representing approx. 3 x 10⁶ clones each.

So far we sequenced and characterised 60 oak cDNA clones, and the identity of 27 clones could be postulated.  This represent 45% of the clones sequenced so far.

One-hundred-thirty Norway spruce cDNA clones have been sequenced.

Seven EST sites showed co-dominant inheritance in the 18 Norway spruce individuals tested.

 

Cathala G et al. (1983) A method for isolation of intact, translationally active ribonucleic acid.  DNA 2(4): 329-339.

Galliano H, Cabane M, Eckerskorn C, Lottspeich F, Sandermann H, Ernst D (1993) Molecular cloning, sequence analysis and elicitor-ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce [Picea abies (L.) Karst.]. Plant Mol. Biol. 23: 145-156.

Gill RW, Hodgman TC, Littler CB, Oxer MD, Montgomery DS, Taylor S, Sanseau P (1997) A new dynamic tool to perform assembly of expressed sequence tags (ESTs). Computer Applications in the Biosciences 13(4): 453-457.

Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270: 467-470.

Table 2: List of the Quercus petraea EST clones.  * Approximate insert length of the cDNA clone established by agarose gel electrophoresis.