generation to the other. This means that the elimination of dioecious male plants
beforeflowering and control of the monoecious state at each generation are nec-
essary procedures to obtain high-quality seed of monoecious cultivars (Beherec
2000 ). The sex phenotype expressed by monoecious plants is subjected to the
influence of genetic factors as well as agronomical practices and environment (Faux
et al. 2013 ); in particular, the involvement of photoperiod suggests that genes
promoting the production of maleflowers by cytologically female plants are also
involved inflowering response to the photoperiod (Faux et al. 2014 ).
C. sativahas a diploid genome (2n = 20) and its karyotype is composed of 9
pairs of autosomes and one pair of sexual chromosomes. Sex determination, in
dioecious plants, seems to be controlled by an X-to-autosome (X/A) balance system
(Shephard et al. 2000 ; Vyskot and Hobza 2004 ), where plants with X/A = 1 are
female, and X/A = 0.5 plants are male (Westergaard 1958 ; Parker and Clark 1991 ;
Ming et al. 2011 ); however, the Y chromosome is reported as essential for normal
pollen development (Shephard et al. 2000 ). Cytological studies have shown the Y
chromosome is larger than X chromosome, subtelocentric, highly heterochromatic
especially in its long arm, and has a satellite portion (Sakamoto et al. 1998 ). The
long arm is rich in several copies of LINE-like retrotransposon repetitive sequences,
which are thought to contribute to the evolutionary differentiation of sex chromo-
somes by partial inhibition of recombination, thus determining the separation of sex
in different individuals (Sakamoto et al. 2000 ; Vyskot and Hobza 2004 ).
A pseudo-autosomal region in the distal part of the euchromatic arm of the Y
chromosome has been identified as carrying several copies of specific subtelomeric
repeats (CS-1), which are also found on both arms of the X chromosome and in the
subtelomeric regions of both arms of all autosomes (Divashuk et al. 2014 ).
Cytogenetic studies on monoecious cultivars indicated the typical diploid
chromosome number (2n = 20) for these plants, but with no Y chromosome and the
presence of two copies of the X chromosome, confirming what had already been
observed by the use of male-specific marker (Mandolino et al. 1999 ;Törjék et al.
2002 ; see below) andflow cytometry studies (Faux et al. 2014 ), and that is that
monoecious hemp has the same karyotype of the dioecious female plants
(Razumova et al. 2015 ).
Different techniques have been employed in the identification of molecular
markers linked to sex in dioecious plants, mainly to male sex, though specific
RAPD markers of female sex have also been described (Shao et al. 2003 , Techen
et al. 2010 ).
As soon asC. sativagenome was analyzed by multi-locus markers such as RAPD
or AFLP, a number of DNA fragments constantly appearing in the male plants but
absent in female and monoecious plants were described. Isolation and sequencing of
MADC1 (Male-Associated DNA Sequence inC. sativa), a 729 bp fragment
obtained by RAPD analysis, (Sakamoto et al. 1995 ), demonstrated that its
transcript-flanking sequences encoded a reverse transcriptase that was homologous
to those belonging to LINE-like retrotransposons from various plants and other
organisms;in situfluorescence hybridization confirmed its localization on telomeric
328 C. Onofri and G. Mandolino