APPROACH
Despite the high economic value of tropical wood, little is known about the genetic control of wood formation or xylogenesis for this species compared to loblolly pine (59,797 ESTs), poplars (25,218 ESTs) and spruce (16,430 ESTs). Wood or secondary xylem is manufactured through the process of cell division, cell expansion, secondary cell wall formation (involving cellulose, hemicellulose, cell wall proteins, and lignin biosynthesis and deposition) and programmed cell death. These processes are strongly interlinked and modulation of any one aspect of wood formation may affect many other aspects.
The use of a functional genomics approach can rapidly provide information on the regulation of not just one gene, but an entire pathway or several pathways at the same time. As of October, 2008, no kelampayan EST information is available in the NCBI GenBank. Therefore, we applied genomics approaches to explore the molecular basis of wood formation in kelampayan via high-throughput DNA sequencing of cDNA clones derived from developing xylem tissues.
Developing xylem tissues were collected by scraping a thin layer from the exposed xylem surface (after removal of the bark) at breast height to investigate the molecular basis of wood formation in kelampayan. A 2-year old tree was sampled at Kota Samarahan, Sarawak in April 2008. The collected tissues were put in a clean plastic bag and immediately frozen in liquid nitrogen in the field, and then kept in -80 °C for later RNA isolation.
Total RNA was extracted using RNeasy Midi Kit (Qiagen, Germany) with modification. Poly(A)+ mRNA was isolated from the total RNA using using Micro-FastTrackTM 2.0 Kits (Invitrogen, USA). A total of 105 µg total RNA was used for mRNA isolation. The purity and quality for both total RNA and mRNA were checked by agarose gel electrophoresis and spectrophotometry.
The cDNA library was constructed using CloneMinerTM cDNA Library Construction Kit (Invitrogen, USA) according to the manufacturer’s protocol. About 0.6 µg mRNA was used as starting template for 1st strand cDNA synthesis. The attB1 adaptor was then ligated at the 5’ end of the double stranded cDNA.
The cDNA was then subjected to size fractionation using cDNA Size Fractionation Columns supplied with the kit. A total of 80 ng size-fractionated cDNA and 250 ng pDONRTM 222 plamid were used for BP recombination reaction. The cDNA was then transformed into ElectroMAXTM DH10B T1 phage resistant cells using MicroPulserTM electroporator (Bio-Rad, USA) and grown on LB-kanamycin agar plates overnight at 37 oC.
cDNA clones were manually picked and cultured overnight with shaking in 96-well culture blocks. Glycerol stocks for each clone were prepared and kept in a duplicate 96-well plate format. All glycerol stocks were kept in -80 °C for later use. The titer of the cDNA library was 1.09 X 107 cfu, indicating that the cDNA library is comprehensive.
A total of 10,368 cDNA clones were randomly selected and used in high-throughput plasmid preparation using Montage Plasmid Miniprep96 and MultiScreen Separation System (Milipore, USA). cDNA inserts were sequenced from the 5’ end using an M13F primer and the ABI PRISMTM Ready Reaction BigDyeTM Terminator Cycler Sequencing kit (Applied Biosystems, USA).
High-throughput DNA sequencing was performed on an ABI 3730xl automated DNA Analyzer (Applied Biosystems, USA). Sequencing and bioinformatics analyses were conducted at the Malaysia Genome Institute (MGI), MOSTI.
All the sequences were quality checked before clustering and annotation. Raw ABI-formatted chromatogram reads were base-called using Phred (Ewing et al., 1998; Ewing and Green, 1998) with a threshold value of 20. Vector sequences were masked using Cross-Match.
The trimming and removal of vectors, adaptors and low quality nucleotides was done using customized Perl scripts. Only high quality ESTs with a minimum of 100 bases and fewer than 4 % N were retained. The high quality ESTs were matched against the NCBI non-redundant database by using the blastx algorithm prior to clustering and assembling of the ESTs.
Sequences with blastx E-value > 10-10 were categorized as having no significant similarity. Multiple sequence alignment, clustering, assembly and the generation of consensus contigs was done using StackPACK (Miller et al., 1999). The StackPACK contains d2_cluster (Burke et al., 1999), PHRAP (Laboratory of PHIL GREEN) and CRAW (Chou and Burke, 1999).
For d2_cluster, the sequences were grouped together if there were at least 96 % sequence similarity in any window of 150 bases. The loose clusters were then aligned using PHRAP and subsequently CRAW. The contigs and singletons generated from the clustering were considered as a set of putative unique genes (unigenes).
References
Burke J, Davison D and Hide W. (1999). d2_cluster: a validated method for clustering EST and full-length cDNA sequences. Genome Res. 9: 1135-1142.
Chou A and Burke J. (1999). CRAWview: for viewing splicing variation, gene families and polymorphism in clusters of ESTs and full-length sequences. Bioinformatics 15: 376-381.
Ewing B, Hillier LAD, Wendl MC and Green P. (1998). Base-calling of automated sequences traces using Phred. Genome Res. 8: 175-185.
Ewing B and Green P. (1998). Base-calling of automated sequences traces using Phred. II. Error probabilities. Genome Res. 8: 186-194.
Laboratory of PHIL GREEN, http://www.phrap.org. (March 24, 2009).
