CRISPR/Cas9-Induced fad2 and rod1 Mutations Stacked With fae1 Confer High Oleic Acid Seed Oil in Pennycress (Thlaspi arvense L.)

Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.

Download Full-text

IDENTIFICATION OF HIGH SEED OIL YIELD AND HIGH OLEIC ACID CONTENT IN BRAZILIAN GERMPLASM OF WINTER SQUASH (Cucurbita moschata D.)

10.1101/2021.07.23.453548 ◽

2021 ◽

Author(s):

Ronaldo Silva Gomes ◽

Ronaldo Machado Júnior ◽

Cleverson Freitas de Almeida ◽

Rebeca Lourenço de Oliveira ◽

Rafael Ravaneli Chagas ◽

...

Keyword(s):

Oleic Acid ◽

Acid Content ◽

Seed Oil ◽

Linoleic Acid Content ◽

Oleic Acid Content ◽

Cucurbita Moschata ◽

High Oleic Acid ◽

Genetic Breeding ◽

High Oleic ◽

High Oleic Acid Content

Cucurbita moschata D. seed oil contains approximately 75% unsaturated fatty acids, with high levels of monounsaturated fatty acids and antioxidant compounds such as vitamin E and carotenoid, constituting a promising food in nutritional terms. Associated to this, the Brazilian germplasm of C. moschata exhibits remarkable variability, representing an important source for the genetic breeding of this vegetable and other cucurbits. In this context, the present study evaluated the productivity and profile of the seed oil of 91 C. moschata accessions from different regions of Brazil and maintained in the Vegetable Germplasm Bank of the Federal University of Viçosa (BGH-UFV). A field experiment was conducted between January and July 2016. The tested C. moschata accessions showed high genetic variability in terms of characteristics related to seed oil productivity (SOP), such as the mass of seeds per fruit and productivity of seeds, providing predicted selection gains of 29.39 g and 0.26 t ha -1 , respectively. Based on the phenotypic and genotypic correlations, greater SOP can be achieved while maintaining high oleic acid content and low linoleic acid content, providing oil of better nutritional and chemical quality. In variability analysis, the accessions were clustered into five groups, which presented different averages for SOP and fatty acid content of seed oil; approach that will guide the use of appropriate germplasm in programs aimed at genetic breeding for SOP and seed oil profile. Per se analysis identified BGH-4610, BGH-5485A, BGH-6590, BGH-5556A, BGH-5472A, and BGH-5544A as the most promising accessions in terms of SOP, with average (m+g) of approximately 0.20 t ha -1 . The most promising accessions for higher oleic acid content of seed oil were BGH-5456A, BGH-3333A, BGH-5361A, BGH-5472A, BGH-5544A, BGH-5453A, and BGH-1749, with average (m+g) of approximately 30%, and almost all of these accessions were also the most promising in terms of lower linoleic acid content of seed oil, with average (m+g) of approximately 45%. Overall, part of the C. moschata accessions evaluated in the present study can serve as a promising resource in genetic breeding programs for SOP and fatty acid profile, aiming at the production of oil with better nutritional and physicochemical quality.

Download Full-text

An insertion of oleate desaturase homologous sequence silences via siRNA the functional gene leading to high oleic acid content in sunflower seed oil

Molecular Genetics and Genomics ◽

10.1007/s00438-008-0391-9 ◽

2008 ◽

Vol 281 (1) ◽

pp. 43-54 ◽

Cited By ~ 32

Author(s):

Séverine Lacombe ◽

Irénée Souyris ◽

André J. Bervillé

Keyword(s):

Oleic Acid ◽

Acid Content ◽

Sunflower Seed ◽

Seed Oil ◽

Homologous Sequence ◽

Oleic Acid Content ◽

High Oleic Acid ◽

High Oleic ◽

Oleate Desaturase ◽

High Oleic Acid Content

Download Full-text

Inheritance of high oleic acid content in the seed oil of soybean mutant M23

Theoretical and Applied Genetics ◽

10.1007/s001220050112 ◽

1996 ◽

Vol 92 (2) ◽

pp. 179-182 ◽

Cited By ~ 5

Author(s):

Y. Takagi ◽

S. M. Rahman

Keyword(s):

Oleic Acid ◽

Acid Content ◽

Seed Oil ◽

Oleic Acid Content ◽

High Oleic Acid ◽

High Oleic ◽

High Oleic Acid Content

Download Full-text

Genetic Control of High Oleic Acid Content in the Seed Oil of Two Soybean Mutants

Crop Science ◽

10.2135/cropsci1996.0011183x003600050009x ◽

1996 ◽

Vol 36 (5) ◽

pp. 1125-1128 ◽

Cited By ~ 19

Author(s):

Shaikh M. Rahman ◽

Yutaka Takagi ◽

Takehito Kinoshita

Keyword(s):

Oleic Acid ◽

Genetic Control ◽

Acid Content ◽

Seed Oil ◽

Oleic Acid Content ◽

High Oleic Acid ◽

High Oleic ◽

High Oleic Acid Content

Download Full-text

Inheritance of high oleic acid content in the seed oil of soybean mutant M23

Theoretical and Applied Genetics ◽

10.1007/bf00223374 ◽

1996 ◽

Vol 92 (2) ◽

pp. 179-182 ◽

Cited By ~ 41

Author(s):

Y. Takagi ◽

S. M. Rahman

Keyword(s):

Oleic Acid ◽

Acid Content ◽

Seed Oil ◽

Oleic Acid Content ◽

High Oleic Acid ◽

High Oleic ◽

High Oleic Acid Content

Download Full-text

Modification of fatty acid composition in soybean seeds to improve soybean oil quality and functionality

10.32469/10355/14220 ◽

2011 ◽

Author(s):

◽

Anh Tung Pham

Keyword(s):

Fatty Acid Composition ◽

Oleic Acid ◽

Fatty Acid ◽

Acid Content ◽

Oil Quality ◽

Soybean Seeds ◽

Oleic Acid Content ◽

High Oleic Acid ◽

Plant Introductions ◽

High Oleic

The purpose of this project is to modify the fatty acid composition in soybean seeds to improve soybean oil quality and functionality. By sequencing the FAD2-1A and FAD2-1B genes in 24 plant introductions, we identified two novel mutant alleles: one for each gene that is responsible for the elevated oleic acid content in four plant introductions. The combination of the newly identified mutant FAD2-1B allele with existing or the novel mutant FAD2-1A alleles created soybean lines with more than 80% oleic acid content. Combination of two mutant FAD2-1A and FAD2-1B with mutant FAD3A or mutant FAD3C or both resulted in high oleic acid content of 80 - 85% and linolenic acid content in the range from 1.5 - 4%. Perfect molecular markers associated with these mutant alleles were designed to help select the soybean lines with genotypes of interest in early generations in breeding. The high oleic acid and high oleic acid low linolenic soybeans produced have an improved stability across growing environments compared to existing sources.

Download Full-text