Use of repetitive sequences to identify DNA polymorphisms linked to regA, a developmentally important locus in Volvox

The regA locus plays a centrally important role in Volvox development by preventing somatic cells from redifferentiating as germ cells; until now, approaches to cloning regA, as a preliminary to molecular analysis of its function, have been lacking. Here a novel approach is described that uses repetitive-sequence probes to rapidly identify restriction fragment length polymorphisms (RFLPs) linked to regA. Genomic DNA was cut with restriction enzymes having 4-base recognition sequences and then electrophoresed long enough to run most fragments off the gel; the remaining long (1- to 20-kb) fragments were resolved into numerous, reproducibly identifiable bands. On Southern blots of such preparations, six repetitive-sequence probes were used to identify 1232 bands, 24% of which were polymorphic between two closely related strains. Ninety-four RFLPs, for which inheritance patterns have been analyzed, fall into 36 "segregation groups," within which no recombination was observed in the limited progeny sample analyzed. Eight RFLPs cosegregated perfectly with alleles at the mating-type (mt) locus. More significantly, four RFLPs exhibited linkage to the regA locus, providing a potential starting place for a chromosome walk designed to clone the locus.