Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system

Abstract

Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.

Figure 1. Chromosomes of C. sativa: C-banding/DAPI, male metaphase (a), male prometaphase (b) and female metaphase (c).

Distribution of Arabidopsis-type (5′-CCCTAAA-3′) telomeric sequences (small red dots located at the end of the chromosomes) (d). FISH with 45S rDNA (green signals) on male (e) and female (f) metaphase chromosomes. Bicolor FISH to male metaphase (g) with CS-1 subtelomeric repeat (red) and 5S rDNA probe (green). FISH to female metaphase (h) with CS-1 subtelomeric repeat (red). The karyotypes of male (i) and female (j) plants. Bar  =  5 µm.

Figure 2. Idiogram of haploid chromosome complement of C. sativa, including Arabidopsis-type telomeric repeat, CS-1 subtelomeric repeat, 45S rDNA and 5S rDNA sites.

Figure 3. The meiotic chromosomes of C. sativa at diakinesis (a) and metaphase I (b).

The chiasma between the sex chromosomes can clearly be seen and indicated by arrows. c Idiogram of the C. sativa XY chromosomes with the hybridization sites of CS-1 (green) and the Arabidopsis-type telomeric repeat (red). The pseudoautosomal region is indicated by brackets.

Figure 4. Simplified phylogeny of Cannabaceae genus included in idiograms.

The phylogeny is according to the reference of . Idiograms created based on data obtained in , , , , and in this study. 5S rDNA: green signals; 45S rDNA: red signals; species-specific subtelomeric repeats (HSR-1for H. lupulus, HJSR for H. japonicus and CS-1 for C. sativa): green signal. The position of pseudoautosomal region on sex chromosomes is indicated by brackets. Time of divergence estimated in , , .