Chromosome Microdissection

Chromosome microdissection, an important tool in genome analysis, has been used to characterize chromosome-specific sequences and to obtain probes for chromosome painting using fluorescent in situ hybridization (FISH) (reviewed in Fominaya et al. 2005).

Unambiguous identification of the chromosome of interest is vital for chromosomal microdissection to be successful. Meiotic cells from introgressed lines containing an alien chromosome of interest can offer unique material for chromosomal microdissection if irregular behavior of the alien chromosome is observed during meiosis (Jung et al. 1992).

Apospory, asexual reproduction through seed, has been transferred to pearl millet from Pennisetum squamulatum through introgression of the ASGR-carrier chromosome via backcrossing (Dujardin and Hanna 1989, J Genet Breed 43:145). Chromosome behavior during meiosis of an advanced backcross generation (BC8) derived from a P. glaucum X P. squamulatum cross was studied. The number of univalents in the BC8 apomict ranged from one to three with one univalent observed in 59% of the meiotic spreads.  The lagging chromosome was almost always the introgressed ASGR-carrier chromosome from P. squamulatum, as determined by FISH analysis using an ASGR-specific bacterial artificial chromosome (BAC) labeled clone. Ten to 15 lagging ASGR-carrier chromosomes were isolated from individual spreads using glass needles and the DNA was amplified using degenerate oligonucleotide primers (DOP).

The amplified DNA was then labeled and compared with the signal generated from a labeled ASGR-specific BAC Both signals co-localized to the same chromosome DNA generated from the ASGR-carrier chromosome has been sequenced (Singh et al. 2010. Characterization of apomictic BC7 and BC8 pearl millet: Meiotic chromosome behavior and construction of an ASGR-carrier chromosome-specific library. Crop Sci. 50:892-902) and used for chromosome painting experiments.

This work was funded by the National Science Foundation (Award 0115911).

 

Peggy Ozias-Akins | The University of Georgia