Abstract
BackgroundTo obtain important expressed sequence tags (ESTs) located on specific chromosomes is currently difficult. Construction of single-chromosome EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes. In this research we developed a method to rapidly isolate ESTs from chromosome 1R of rye by combining the techniques of chromosome microdissection with hybrid specific amplification (HSA).ResultsChromosome 1R was isolated by a glass needle and digested with proteinase K (PK). The DNA of chromosome 1R was amplified by two rounds of PCR using a degenerated oligonucleotide 6-MW sequence with a Sau3AI digestion site as the primer. The PCR product was digested with Sau3AI and linked with adaptor HSA1, then hybridized with the Sau3AI digested cDNA with adaptor HSA2 of rye leaves with and without salicylic acid (SA) treatment, respectively. The hybridized DNA fragments were recovered by the HSA method and cloned into pMD18-T vector. The cloned inserts were released by PCR using the partial sequences in HSA1 and HSA2 as the primers and then sequenced. Of the 94 ESTs obtained and analyzed, 6 were known sequences located on rye chromosome 1R or on homologous group 1 chromosomes of wheat; all of them were highly homologous with ESTs of wheat, barley and/or other plants in Gramineae, some of which were induced by abiotic or biotic stresses. Isolated in this research were 22 ESTs with unknown functions, probably representing some new genes on rye chromosome 1R.ConclusionWe developed a new method to rapidly clone chromosome-specific ESTs from chromosome 1R of rye. The information reported here should be useful for cloning and investigating the new genes found on chromosome 1R.
Highlights
To obtain important expressed sequence tags (ESTs) located on specific chromosomes is currently difficult
The strategy of this method is the combination of chromosome microdissection method and hybrid specific amplification (HSA) technique [36] to yield the homologous sequences between microdissected DNA and cDNA
The adaptors and primers described above are designed from suppression subtractive hybridization (SSH) [37], with a change of the blunt ends into annealing ends of Sau3AI
Summary
To obtain important expressed sequence tags (ESTs) located on specific chromosomes is currently difficult. Construction of single-chromosome EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes. Large-scale mapping of EST unique genes can provide valuable insights into the organization of genomes and chromosomes [8]. 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 for large genome species such as wheat [8,9,10]. 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. ESTs of microdissected chromosomes had been isolated successfully by using microdissection-mediated cDNA capture [13,14]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
