ARS | Publication request: Isolation of Expressed Sequences from a Specific Chromosome of Thinopyrum Intermedium Infected by BYDV

Submitted to: Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2008
Publication Date: December 18, 2008
Citation: Jiang, S.M., Yin, W.B., Hu, J., Shi, R., Zhou, R.N., Chen, Y.H., Zhou, G.H., Wang, R., Song, L.Y., Hu, Z.M. 2008. Isolation of Expressed Sequences from a Specific Chromosome of Thinopyrum Intermedium Infected by BYDV. Genome 52:68-76.

Interpretive Summary: Expressed sequence tag (EST) analysis is one of ways to clone genes controlling desirable traits in plant. At present, most of ESTs registered in GenBank databases are from cDNA libraries constructed out of different tissues at different developmental stages. EST identification and mapping are laborious and time consuming, especially for polyploid plants. Although many important genes have been located on specific chromosome and/or specific regions of chromosomes by traditional genetics and modern molecular biotechniques, isolating expressed sequences directly from specific chromosomes or specific chromosome segments would be more advantageous. BYDV causes serious yield losses in all cereals worldwide. Resistance genes against BYDV have not been found in common wheat but available in barley, oat and some wild Gramineae species. Until now, no resistance genes against BYDV were cloned, but several polypeptide markers and PCR markers linked with BYDV resistance gene have been reported. We modified the techniques to isolate the expressed sequences from an intermediate wheatgrass chromosome that resulted in the cloning of 12 expressed sequences from the alien chromosome in TAi-27 following BYDV infection. It is an improved method for genomic and functional genomics research of polyploid plants.

Technical Abstract: To map important ESTs to specific chromosomes and/or chromosomal regions is difficult in hexaploid wheat because of its large genome size and serious interference of homologous sequences. Large scale EST sequencing and subsequent chromosome localization are both laborious and time-consuming. The wheat alien addition line TAi-27 contains a pair of chromosomes of Thinopyrum intermedium that carries the resistance gene against barley yellow dwarf virus (BYDV). In this research, we developed a modified technique based on chromosome microdissection and hybridization specific amplification (HSA) to isolate expressed sequences from the alien chromosome of TAi-27 by hybridization between the DNA of microdissected alien chromosome and cDNA of Thinopyrum intermedium infected by BYDV. Twelve clones were selected, sequenced and analyzed. Three of them are unknown genes without any hit in the GenBank database, and the other nine are highly homologous with ESTs of wheat, barley and/or other plants in Gramineae induced by abiotic or biotic stress. The method used in this research to isolate expressed sequences from a specific chromosome has following advantages: 1) the obtained expressed sequences are larger in size and have 3' end information; 2) the operation is less complicated. It would be an efficient improved method for genomics and functional genomics research of polyploid plants, especially for ESTs development and mapping. The obtained expressed sequence data are also informative to understand the resistant genes on the alien chromosome of TAi-27.