Barley is one of the world's most important cereal crops used for food, malt and animal feed.
However it has a relatively large genome size (5 Gbp)
which inhibits full-scale analysis of the genome structure by sequencing.
An alternative way to understanding the structure and function of the barley genes is
by full-length cDNA approach.
From 2005, the cDNA libraries of malting barley ( Hordeum vulgare cv. Haruna Nijo)
using the "cap-trapper" method have been constructed as part of the Green-Techno Program led by Japanese MAFF.
The conditions for preparation of RNA materials for cDNA construction are as follows. Each cDNA sequence is identified for its origin of tissue by the 8-base sequence tag identifier listed below.
|Library||Tag No.||Tissue||Brevity code||Tag sequence||Tag comlement|
|N library||1-1||Flag leaf||FGL||TACGTACG||CGTACGTA|
|1-2||Cold treatment shoot||CTS||CATGTACG||CGTACATG|
|1-3||Haruna normal shoot
(Shaded or water culture)
|S library||2-1||Hydroponic aluminium treated plant||HAP||CTGACATG||CATGTCAG|
|2-2||Hydroponic drought stress or salt stress||HDS||TACGCATG||CATGCGTA|
|2-3||ABA treatment plant||ABP||GTCACATG||CATGTGAC|
|2-4||JA treatment plant||JAP||CTGACTGA||TCAGTCAG||PMF library||3-1||Young spike panicle||YSP||TACGCTGA||TCAGCGTA|
|3-2||Flower in heading||FLH||CATGCTGA||TCAGCATG|
|3-3||Flower in maturing||FLM||GTCACTGA||TCAGTGAC|
After construction of library, 96 clones of each library were picked randomly and measure the length of insert DNA to verify quality of these libraries. cDNA clones were cut by PvuII and load in 1.5% agarose gel with 500 bp ladder marker. The insert lengths of 3 pooled libraries were 1.5-1.7 kb and all three libraries were considered reasonable as the length of plant full length cDNA library. 172,800 clones (57,600 sequences for each library) were sequenced both end with BDT v3.1 and ABI3700 and check read length with Phred. The read length in this study was more than 500 bp and considered reasonable. 567 clones of representative clones had homolog of TEs or chloroplast, so these clones were removed from list of full-length sequencing. The 3' end sequences were clustered independently based on BLAST analysis of end sequences and clones with the most 5' proximal sequences in each group are selected as representative clones and being used for full-length sequencing with primer-walking. In this database, all the 5' and 3' end sequences from the libraries are used for sequence clustering, and the resulted contigs are presented for further investigation.