Submitted to: Biomed Central (BMC) Plant Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/1/2008
Publication Date: 6/3/2008
Citation: Zhou, R-N., Shi, R., Jiang, S-M., Yin, W-B., Wang, H-H., Chen, Y-H., Hu, J., Wang, R. R.-C., Zhang, X-Q., Hu, Z-M. 2008. Rapid EST isolation from chromosome 1R of rye. BMC Plant Biology doi:10.1186/1471-2229-8-28. Interpretive Summary: Expressed sequence tag (EST) analysis has opened up exciting prospects for gene discovery in all organisms, irrespective of their genome size. Large-scale mapping of EST unique genes can provide valuable insights into the organization of genomes and chromosomes. EST distribution in relation to chromosome landmarks (short and long arms, euchromatin, heterochromatin, centromeres, and telomeres) and recombination is important in comparative analysis of chromosome structure and evolution, gene isolation, and targeted genome sequencing. Construction of single chromosome or chromosome region EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes and/or specific chromosomal regions. Rye (Secale cereale L., 2n=14, genome R) has good adaptability to extreme climatic and soil conditions, requiring low amounts of fertilizers and pesticides. Chromosome 1R carries genes for resistance to powdery mildew, stem rust, leaf rust, yellow rust, and greenbug. In this research, we developed a new method to rapidly isolate ESTs of chromosome 1R from rye leaves by using the techniques of chromosome microdissection combined with hybrid specific amplification (HSA). Twenty-two (22) ESTs with unknown functions probably represent some new genes on rye chromosome 1R.
Technical Abstract: To obtain important expressed sequence tags (ESTs) located on specific chromosomes is difficult at present. Construction of single chromosome EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes. In this research, we develop a method to rapidly isolate ESTs from chromosome 1R of rye by combining the techniques of chromosome microdissection with hybrid specific amplification (HSA). The chromosome 1R was isolated by a glass needle and digested with proteinase K (PK). The DNA of the chromosome 1R was amplified by two rounds of PCR using degenerated oligonucleotide 6-MW sequence with a Sau3AI digestion site as the primer. The PCR product was digested with SauAI and linked with adaptor HSA1 then hybridized with the Sau3AI digested cDNA with adaptor HSA2 of rye leaves with and without treatment of salicylic acid (SA), respectively. The hybridized DNA fragments were recovered by the HSA method and cloned into pMD18-T Vector. The inserts were released by PCR using the partial sequences in HSA1 and HSA2 as the primers and then sequenced. Ninety-four (94) ESTs were obtained and analyzed. Compared with sequences in GenBank database, 6 of them were known sequences located on rye chromosome 1R or homologous group 1 chromosomes of wheat; all of them were highly homologous with ESTs of wheat, barely and/or other plants in Gramineae; and some of which were induced by abiotic or biotic stresses. Isolated in this research are 22 ESTs with unknown functions, probably representing some new genes on rye chromosome 1R. We developed a new method to rapidly clone chromosome specific ESTs. It would be a useful method to investigate genes on a specific chromosome. Furthermore, the information reported here should be useful to clone and investigate the new genes on chromosome 1R.