regions of the Y chromosome (Sakamoto et al. 2000 ), giving thefirst hints about the
structure of sexual chromosomes inCannabis.
Further male-associated sequences were identified and used as male-specific
markers inC. sativa; a 390 bp fragment identified by RAPD analysis showed the
existence of another sequence (MADC2) linked to male sex in dioecious hemp
(Fig.15.1d). The marker sequence had limited (50–60%) homology with other
plant sequences belonging to repeated regions or retrotransposon-like sequences,
and had no open reading frames, most probably belonging to a non-coding genome
region of the Y chromosome. The sequencing of the fragment allowed the devel-
opment of a SCAR marker (Mandolino et al. 1999 ), which demonstrated full
association with the male sex.
More comprehensive studies were performed by Mandolino et al. ( 2002 ) and
Sakamoto et al. ( 2005 ), with a screening of RAPD markers that led to the identi-
fication of 10 and 17 new male-associated markers, respectively. Such markers,
when used on F1 progenies, never showed breaking the association with sex
(Mandolino et al. 2002 ), suggesting that male-associated markers were located on
the portion of Y chromosome excluded from the recombination during meiosis.
MADC3 and MADC4 sequences (Sakamoto et al. 2005 ), 771 bp- and 576-bp
long respectively, upon use asfluorescent probes for in situ hybridization studies,
showed their localization either on Y chromosome and on all autosomes (MADC3),
or specifically on Y and on one pair of autosomes (MADC4). The MADC3
sequence revealed the presence of a coding region that is highly homologous to
open reading frames, which encode the gag/pol polyprotein of copia-like retro-
transposons in various plant species, and of a second coding region at the 5’-end
that is homologous to RNAse H, whereas the MADC4 sequence showed a simi-
larity, on the aminoacid level, with the integrase in the polyprotein of the copia-like
retrotransposon from rice (Sakamoto et al. 2005 ). Many other male-associated
markers were developed by Flachowsky et al. ( 2001 ) through AFLP analysis and
Bulk Segregant Analysis of both male- and female-plant DNA bulks and segre-
gating progenies belonging to two different hemp accessions: eleven male-specific
fragments were detected, and again, a lack of recombination events for all the
male-specific AFLP markers was observed, suggesting their strict co-localization
with male sex locus.
However, a detailed analysis of different marker classes observed in the pro-
genies led to the definition of the classical male-associated markers putatively
present only on Y chromosome and in regions excluded by recombination (class E
markers in Peil et al. 2003 and in Faux et al. 2016 ), in addition to markers
heterozygous in both parents, and located on one X chromosome of the female
parent and on the Y chromosome of the male parent (class D markers). These
markers were interpreted as being associated to a chromosome region where
recombination between the two sexual chromosomes occurs, called Pseudo
Autosomal Region (Peil et al. 2003 ) and already cytogenetically demonstrated in
Cannabisat meiotic prophase I in pollen mother cells (Sakamoto et al. 2000 ). The
male-associated markers, therefore, besides their application inC. sativabreeding,
15 Genomics and Molecular Markers inCannabis sativaL. 329