Potentials and Challenges of Genomics for Breeding Cannabis Cultivars

Abstract

Cannabis (Cannabis sativa L.) is an influential yet controversial agricultural plant with a very long and prominent history of recreational, medicinal, and industrial usages. Given the importance of this species, we deepened some of the main challenges-along with potential solutions-behind the breeding of new cannabis cultivars. One of the main issues that should be fixed before starting new breeding programs is the uncertain taxonomic classification of the two main taxa (e.g., indica and sativa) of the Cannabis genus. We tried therefore to examine this topic from a molecular perspective through the use of DNA barcoding. Our findings seem to support a unique species system (C. sativa) based on two subspecies: C. sativa subsp. sativa and C. sativa subsp. indica. The second key issue in a breeding program is related to the dioecy behavior of this species and to the comprehension of those molecular mechanisms underlying flower development, the main cannabis product. Given the role of MADS box genes in flower identity, we analyzed and reorganized all the genomic and transcriptomic data available for homeotic genes, trying to decipher the applicability of the ABCDE model in Cannabis. Finally, reviewing the limits of the conventional breeding methods traditionally applied for developing new varieties, we proposed a new breeding scheme for the constitution of F₁ hybrids, without ignoring the indisputable contribution offered by genomics. In this sense, in parallel, we resumed the main advances in the genomic field of this species and, ascertained the lack of a robust set of SNP markers, provided a discriminant and polymorphic panel of SSR markers as a valuable tool for future marker assisted breeding programs.

Keywords: Cannabis sativa, breeding methods; DNA barcodes; F1 hybrids; MADS box genes; simple sequence repeat markers.

Figure 1

Information on sex determinants (A) and sex chromosomes (B) in cannabis [adapted from (Bergero and Charlesworth, 2008; Divashuk et al., 2014)].

Figure 2

Method used for the development of the “Skunk No. 1”: the first NLD/BLD hybrid bred in the early 1970s. To obtain this variety, plants of the F₂ progeny were chosen to carry out nine repeated inbreeding cycles aimed at increasing their homozygosity, then ten female and ten male plants were selected and vegetatively propagated to be used as parental lines in all possible pairwise cross-combinations.

Figure 3

(A) Similarity-based neighbor-joining analysis performed using 21 amino acid sequences from the C. sativa (Cs) proteome (GCA_900626175.1) selected for their putative orthology (Table 2 and, more specifically, Supplementary Table 2) with well-characterized ABCDE MADS box proteins belonging to Arabidopsis thaliana (At) and Vitis vinifera (Vvi). (B) Taking advantage of a recent in silico analysis of 31 RNA-seq datasets derived from different tissues of two different psychoactive strains (Finola and Purple Kush, NCBI SRA accession numbers: SRP006678 and SRP008673) of C. sativa (Massimino, 2017), a principal component analysis was performed using the expression values of the MADS box genes previously identified. The analysis is based on the ln(x+1) transformed (RPKM) values (reads per kilobase of transcript per million mapped reads) and showed a clear separation of samples related to reproductive organs from those related to vegetative organs. (C) Heat map showing the relative expression of each gene in the different tissues considered.

Figure 4

Breeding methods for the development of commercial F₁ hybrid cultivars: two-way (A), three-way (B) and four-way (C) F₁ hybrids with inbreeding progression in case of selfing and full-sibling crosses (D) and large-scale hybridization and F₁ female-seed production (E).

Figure 5

Information on SSR regions. (A) Abundance of the main repeat types (% base pairs among the total base pairs of the motifs) of SSR sites in the Cannabis cs10 genome. (B) Abundance of the motifs at the total SSR sites.

Figure 6

Individual linkage groups in the Cannabis genome (n = x = 10) with the physical position and genetic information of the selected SSR markers. Basic information on intergene and intragene sites, including intron/exon positions of SSR markers, and their corresponding physically linked genes are also reported (marker loci found to be polymorphic among all the three explored genomes are marked with an asterisk).