Introduction
Alfalfa is an open-pollinated autotetraploid (2n=4x= 32)
that shows extensive genetic variability within its gene pool
(Jones and Bingham 1995). Cultivars currently available
are not well adapted to harsh winter conditions that occur in
the midwest United States, Canada and northern Europe
(Volenec et al. 2002). Improvement of winter hardiness has
essentially been achieved by selection within Weld nurseries
of plants that survive test winters. However, the unpredictability of the occurrence of adequate screening conditions
along with genotype by environment (G£E) interactions
requires the costly maintenance of nurseries for many years
and, in some cases, at several locations (Limin and Fowler
1991). We recently applied a recurrent selection protocol
entirely performed indoor to accelerate the development of
alfalfa populations selectively improved for their tolerance
to freezing (TF populations). These TF populations showed
signiWcant increases in freezing tolerance and survival to
harsh winter conditions in response to selection (Castonguay et al. 2009).
Bulked segregant analysis (BSA; Michelmore et al.
1991) is a simple and very eVective approach to identify
genetic polymorphisms linked to a speciWc trait by comparing pools of DNA from plant siblings with contrasted