Identification of Expressed Sequence Markers for a Major Gene-Rich Region of Wheat Chromosome Group 1 Using RNA Fingerprinting–Differential Display
2002
Sandhu, Devinder | Sidhu, Deepak | Gill, Kulvinder S.
This study demonstrates a successful application of RNA fingerprinting–differential display technique in identifying expressed sequence markers for a small targeted region of the wheat (Triticum aestivum L.) genome. Wheat genes are present in clusters spanning about 10% of the genome. One of the important gene-rich regions is present on the short arm of wheat homoeologous group 1 chromosomes around fraction length 0.8 (‘1S0.8 region’). The region is about 0.1% of the wheat genome and is flanked by the breakpoints of deletion lines 1BS-4 and 1BS-19. The objective of this study was to identify expressed sequence markers for the region. First-strand cDNA of poly A⁺ mRNA pooled from various developmental stages of the two deletion lines were PCR amplified in the presence of ³⁵S by means of 90 pair-wise combinations of 19 primers. Amplification products were size-separated on a denaturing polyacrylamide urea gel. A total of 6840 fragment bands were amplified, of which 65 were present in the deletion line 1BS-4, but missing in 1BS-19. These 65 fragment bands were cut out of the gel, reamplified, and used as probes for gel-blot DNA analysis of group 1 nullisomic-tetrasomic lines and the two deletion lines. Nineteen of the 65 fragment bands detected a smear pattern and thus were not mapped. Of the remaining 46 probes, 22 mapped to wheat homoeologous group 1 and seven mapped to the ‘1S0.8 region’. The same approach can be used to target other wheat gene-rich regions bracketed by deletion breakpoints.
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