Plant & Animal Genomes XI Conference

January 11-15, 2003
Town & Country Convention Center
San Diego, CA

Poster: Genome Sequencing & ESTs
            

CONSTRUTION OF FULL LENGTH-ENRICHED CDNA LIBRARY IN BARLEY

¹ Research Institute for Bioresources, Okayama Univ., Kurashiki, 710-0046, Japan

² Core Research Evolutional Science and Technology, Japan Science and Technology Corporation, Japan

³ National Institute of Genetics, Mishima, 411-8540, Japan

Using the "oligo-capped" mRNA method (Maruyama and Sugano 1994), we constructed cDNA libraries in barley. They are "full length-enriched cDNA libraries" which have a high content of full-length cDNA clones. Leaves from etiolated seedlings (2-leaf stage) (ET) and shoots from germinating stage (5-day old) (ST) of malting barley "Haruna Nijo" were used as sources of mRNA. Average insert sizes were 1.2kb (ST) and 1.5kb (ET), respectively. For ST, we size fractioned the cDNA more than 3kb (STL). In order to estimate the level of full-length cDNA content in each library, we performed one-pass sequencing of 96 randomly selected cDNA clones from the 5' end in June 2001 (ET) and January 2002 (ST and STL), respectively. By blastn analysis, frequencies of possible full-length cDNA clones, whose sequence had the known 5' ends or sequences upstream of the known 5' ends, were 78% (ET), 50% (ST) and 94% (STL), respectively, although many of the sequences did not hit with public DNA sequences. Further we have randomly sequenced the cDNA clones in each library from both the 5' and 3' ends (no. of clones, ET:3604, ST:3478, STL:2050). However, the same blastn analysis procedure in October 2002 produced a much lower frequency of full-lengh cDNA content (ET:31%, ST:21%, STL:34%). This can be attributed to the abundance of sequences such as rice BACs after the initial analyses. A more precise estimation may be possible after analysing rice full-length cDNA sequences from the Japanese Rice Genome Project, which will be available in the near future.