National BioResource Project of Japan: DNA resource of Wheat

Yasunari Ogihara

Kihara Institute for Biological Research, Yokohama City University

Maioka-cho 641-12, Yokohama 244-0813, Japan

Corresponding author: Yasunari Ogihara

E-mail: yogihara@yokohama-cu.ac.jp

Wheat is characteristic of its polyploid nature.  Mainly because of its huge genome size (5Gbp per genome), information of entire genome sequencing is not sufficient.  In order to carry out functional genomics in common wheat, we had made a plan to complete expressed sequence tags (ESTs) in common wheat mainly using Chinese Spring for construction of gene expression map in various tissues of wheat and supplying new marker information for the chromosome mapping.  At first, we extracted total RNAs from 11 tissues during the wheat life cycle.  The cDNA libraries with plasmid had been constructed from these RNAs without any amplification, so that the cDNA libraries should be reflected their mRNA abundances.  After several thousands bacterial colonies from each library were randomly picked up, one pass sequencing had been carried out from both ends of each insert DNA.  These ESTs were classified into certain contigs with the phrap method.  We were able to distinguish each contig corresponding to their homoeologues from three genomes, namely A, B and D by adjusting the parameter of the phrap.  By counting the number of ESTs in each contig, we displayed gene expression patterns in their tissues.  We called this the “Virtual Display: VD”.  VD allows us to trace the global gene expression profiles of interest during the wheat life cycle, and is going to be opened.  Then we collected further RNAs from the stressed tissues such as drought, salt, cold/heat shock, cultures, metals, and disease fungi as well as additional tissues during the life cycle.  In total, gene expression patterns from the 56 tissues and/or stress-treatments, at present can be displayed.  More than 630 thousands wheat EST data are available, and these ESTs were classified into ca. 38 thousands gene clusters.  Since total gene numbers were predicted to be ca. 40 thousands from the entire sequencing of rice genome, more than 90% of genes can be captured even in wheat.  By applying these cDNA sequence data, we constructed the agilent oligo DNA microarray harboring 38K gene probes.  The wheat oligo DNA microarray is now available from our lab (yogihara@yokohama-cu.ac.jp).   In addition to ordinary cDNA libraries of common wheat, we had constructed the wheat full length cDNA library.  We extracted total RNAs from 13 tissues and/or stress-treatments.  These RNAs were mixed, and supplied for construction of the full length cDNA library with the CAP-trapper method.  Approximately 20,000 clones were picked up to carry out one pass sequencing from both ends of inserts.  By grouping these cDNA sequences, 6162 clones were selected to complete the entire sequencing of inserts.

Furthermore, we had constructed the genomic library of Chinese Spring wheat with the transformation-competent artificial chromosome (TAC vector).  This TAC library covers ca. three times of the Chinese Spring wheat genome to be proven the selection of single copy gene from the library.  In response to user’s request, we selected positive clones against the special targets.

These lines of activities indicate that DNA resources of common wheat are substantial and useful, and are required to complete their line-ups in the next stage (Fig. 1).