A high-throughput segregation analysis identifies the sex chromosomes ofCannabis sativa

Abstract

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).

Sequence and annotation of 42 cannabis genomes reveals extensive copy number variation in cannabinoid synthesis and pathogen resistance genes

“…These male-specific mRNAs were then intersected with the male-specific contigs to identify 574 genes on the non-recombining region of the Y chromosome. Prentout et al has reported a similar approach using Illumina based RNAseq with Type I cannabis plants but the data is not currently available (Prentout 2019). It is important to note that reads that do not map to the female reference can be either 1) the Y chromosome and or 2) a structural variation (SVs) in the female reference genome.…”

“…To confirm the 118Mb list of putative Y contigs, we utilized Illumina’s NovaSeq platform to whole genome sequence 40 hemp and drug-type cultivars. Using the coverage maps of 9 male genomes, we were able to confirm putative Y contigs and male-specific genes (Prentout 2019). We further classified these variants to identify highly damaging mutations in protein coding regions of the genome.…”

The wild grape genome sequence provides insights into the transition from dioecy to hermaphroditism during grape domestication

“…To identify sex-linked polymorphisms in V. sylvestris, we produced a cross and resequenced in paired-end Illumina short reads the whole genomes of the parents and 10 offspring, yielding 43-74 millions of read pairs per individual (Supplementary Table 2). To overcome issues due to X-Y divergence, we used an iterative SNP-tolerant mapping procedure 11 , eventually mapping 98.2% of reads in average (mapping coverage by individual 14x-28x, Supplementary Table 3-6). Single nucleotide polymorphisms (SNPs) that segregate with sex in our cross were identified with an empirical approach and with SEX-DETector ++ , a new version of the probabilistic method SEX-DETector that identifies sex-linked genes from patterns of segregation in a cross from RNA-seq data 12 , which we developed here to analyse genomic data.…”

“…Divergence between X and Y alleles can prevent SNP discovery. To avoid this issue, we carried out an iterative SNP-tolerant mapping similarly to the procedure described in Prentout et al 11 . Raw reads of twelve individuals of the same family (2 parents and 5 descendants of each sex) were first mapped against the V. sylvestris genome with gsnap 42 v2018-07-04 (-m 0.1) in standard mode.…”

Detecting sex-linked genes using genotyped individuals sampled in natural populations

“…Recently, the SEX-DETector statistical framework, relying on the modeling of the transmission of alleles that account for the observed genotypes, was introduced (Muyle et al, 2016). The method has been shown to yield high power and high sensitivity with as few as five offspring of each sex, and has successfully been used on a number of species (Muyle et al, 2018;Veltsos et al, 2019;Martin et al, 2019;Prentout et al, 2020). It can be used for several purposes, using a single dataset: testing whether a species has sex chromosomes and of which kind (XY or ZW), identifying sex-linked genes, reconstructing the haplotypes of the gametolog copies, and estimating expression of each of the copies if RNAseq data are used.…”