A high-throughput segregation analysis identifies the sex chromosomes of Cannabis sativa

Cannabis sativa-derived tetrahydrocannabinol (THC) production is increasing very fast worldwide. C. sativa is a dioecious plant with XY chromosomes, and only females (XX) are useful for THC production. The C. sativa sex chromosomes sequence would improve early sexing and better management of this crop; however, the C. sativa genome projects failed to identify the sex chromosomes so far. Moreover, dioecy in the Cannabaceae family is ancestral, C. sativa sex chromosomes are potentially old and thus very interesting to study as little is known about the last steps of sex chromosome evolution in plants. Here we RNA-sequenced a C. sativa family (2 parents and 10 male and female offspring) and performed a segregation analysis for all C. sativa genes using the probabilistic method SEX-DETector. We identified >500 sex-linked genes. Mapping of these sex-linked genes to a C. sativa genome assembly identified a single chromosome pair with a large non-recombining region. Further analysis of the >500 sex-linked genes revealed that C. sativa has a strongly degenerated Y chromosome and represents the oldest plant sex chromosome system documented so far. Our study revealed that old plant sex chromosomes can have large non-recombining regions and be very differentiated and still be of similar size (homomorphic).