Targeted mutation of Δ12 and Δ15 desaturase genes in hemp produce major alterations in seed fatty acid composition including a high oleic hemp oil

Abstract

SummaryWe used expressed sequence tag library and whole genome sequence mining to identify a suite of putative desaturase genes representing the four main activities required for production of polyunsaturated fatty acids in hemp seed oil. Phylogenetic-based classification and developing seed transcriptome analysis informed selection for further analysis of one of seven D12 desaturases and one of three D15 desaturases that we designate CSFAD2A and CSFAD3A, respectively. Heterologous expression of corresponding cDNAs in Saccharomyces cerevisiae showed CSFAD2A to have Dx+3 activity, while CSFAD3A activity was exclusively at the D15 position. TILLING of an ethyl methane sulphonate mutagenized population identified multiple alleles including non-sense mutations in both genes and fatty acid composition of seed oil confirmed these to be the major D12 and D15 desaturases in developing hemp seed. Following four backcrosses and sibling crosses to achieve homozygosity, csfad2a-1 was grown in the field and found to produce a 70 molar per cent high oleic acid (18:1 D9 ) oil at yields similar to wild type. Cold-pressed high oleic oil produced fewer volatiles and had a sevenfold increase in shelf life compared to wild type. Two low abundance octadecadienoic acids, 18:2 D6,9 and 18:2 D9,15 , were identified in the high oleic oil, and their presence suggests remaining endogenous desaturase activities utilize the increased levels of oleic acid as substrate. Consistent with this, CSFAD3A produces 18:2 D9,15 from endogenous 18:1 D9 when expressed in S. cerevisiae. This work lays the foundation for the development of additional novel oil varieties in this multipurpose low input crop.

The Complex Interactions Between Flowering Behavior and Fiber Quality in Hemp

“…Large populations of mutated plants are then screened for the presence of mutations in specific genes using high throughput sequencing, or other screening technologies. This strategy was used in hemp to find plants with specific induced knock-out and missense mutations in CsFAD2 and CsFAD3 genes leading to altered seed-oil composition in the seed hemp variety Finola (Bielecka et al., 2014). Such a strategy requires facilities to grow large mutant populations and for seed storage and breeding steps to obtain homozygous mutations or combine different mutations.…”

In silico gene expression profiling in Cannabis sativa

“…One potential candidate is the Csfad2a gene which was recently found to be highly expressed only in some hemp strains. Here, high Csfad2a expression was correlated with both higher oil content and lower oxidation tendency, eventually leading to the production of a significantly better commercial product 26 .…”

Cannabis, from plant to pill

“…A mutagenesis strategy employing ethyl methanesulfonate (EMS) has been successfully utilized to modify the seed oil profile of industrial hemp , indicating the possible success of a reverse genetics strategy to manipulate the cannabinoid and terpenoid profiles of Cannabis . However, as C. sativa is predominantly a dioecious species, although a small number of hemp accessions or varieties are monoecious, the path towards the generation of a successful commercial variety can be somewhat protracted, as it relies upon the crossing of female lines harbouring superior traits with elite male siblings.…”