TILLING-by-Sequencing+ Reveals the Role of Novel Fatty Acid Desaturases (GmFAD2-2s) in Increasing Soybean Seed Oleic Acid Content

Soybean is the second largest source of oil worldwide. Developing soybean varieties with high levels of oleic acid is a primary goal of the soybean breeders and industry. Edible oils containing high level of oleic acid and low level of linoleic acid are considered with higher oxidative stability and can be used as a natural antioxidant in food stability. All developed high oleic acid soybeans carry two alleles; GmFAD2-1A and GmFAD2-1B. However, when planted in cold soil, a possible reduction in seed germination was reported when high seed oleic acid derived from GmFAD2-1 alleles were used. Besides the soybean fatty acid desaturase (GmFAD2-1) subfamily, the GmFAD2-2 subfamily is composed of five members, including GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E. Segmental duplication of GmFAD2-1A/GmFAD2-1B, GmFAD2-2A/GmFAD2-2C, GmFAD2-2A/GmFAD2-2D, and GmFAD2-2D/GmFAD2-2C have occurred about 10.65, 27.04, 100.81, and 106.55 Mya, respectively. Using TILLING-by-Sequencing+ technology, we successfully identified 12, 8, 10, 9, and 19 EMS mutants at the GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E genes, respectively. Functional analyses of newly identified mutants revealed unprecedented role of the five GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E members in controlling the seed oleic acid content. Most importantly, unlike GmFAD2-1 members, subcellular localization revealed that members of the GmFAD2-2 subfamily showed a cytoplasmic localization, which may suggest the presence of an alternative fatty acid desaturase pathway in soybean for converting oleic acid content without substantially altering the traditional plastidial/ER fatty acid production.

A TILLING by sequencing approach to identify induced mutations in sunflower genes

The Targeting Induced Local Lesions in Genomes (TILLING) technology is a reverse genetic strategy broadly applicable to every kind of genome and represents an attractive tool for functional genomic and agronomic applications. It consists of chemical random mutagenesis followed by high-throughput screening of point mutations in targeted genomic regions. Although multiple methods for mutation discovery in amplicons have been described, next-generation sequencing (NGS) is the tool of choice for mutation detection because it quickly allows for the analysis of a large number of amplicons. The aim of the present work was to screen a previously generated sunflower TILLING population and identify alterations in genes involved in several important and complex physiological processes. Twenty-one candidate sunflower genes were chosen as targets for the screening. The TILLING by sequencing strategy allowed us to identify multiple mutations in selected genes and we subsequently validated 16 mutations in 11 different genes through Sanger sequencing. In addition to addressing challenges posed by outcrossing, our detection and validation of mutations in multiple regulatory loci highlights the importance of this sunflower population as a genetic resource.

TILLING-by-Sequencing+ to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis

Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing+ (TbyS+), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS+ in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, GmSACPD, GmFAD2, and GmFAD3, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS+, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.

Improving soybean seed oil without poor agronomics

This article comments on: Lakhssassi N, Zhou Z, Liu S, Piya S, Cullen MA, El Baze A, Knizia D, Patil GB, Badad O, Embaby MG, Meksem J, Lakhssassi A, Ghazaleh A, Hewezi T, Meksem K. 2020. Soybean TILLING-by-sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules. Journal of Experimental Botany 71, 6969–6987.

Soybean TILLING-by-Sequencing+ reveals the role of novel GmSACPD members in unsaturated fatty acid biosynthesis while maintaining healthy nodules

Developing soybean lines with high levels of stearic acid is a primary goal of the soybean industry. Most high-stearic-acid soybeans carry different GmSACPD-C mutated alleles. However, due to the dual role of GmSACPD-C in seeds and nodule development, all derived deleterious GmSACPD-C mutant alleles are of extremely poor agronomic value because of defective nodulation. The soybean stearoyl-acyl carrier protein desaturase (GmSACPD) gene family is composed of five members. Comparative genomics analysis indicated that SACPD genes were duplicated and derived from a common ancestor that is still present in chlorophytic algae. Synteny analysis showed the presence of segment duplications between GmSACPD-A/GmSACPD-B, and GmSACPD-C/GmSACPD-D. GmSACPD-E was not contained in any duplicated segment and may be the result of tandem duplication. We developed a TILLING by Target Capture Sequencing (Tilling-by-Sequencing+) technology, a versatile extension of the conventional TILLING by sequencing, and successfully identified 12, 14, and 18 ethyl methanesulfonate mutants at the GmSACPD-A, GmSACPD-B, and GmSACPD-D genes, respectively. Functional analysis of all identified mutants revealed an unprecedented role of GmSACPD-A, GmSACPD-B, and GmSACPD-D in unsaturated fatty acid biosynthesis without affecting nodule development and structure. This discovery will positively impact the development of high-stearic-acid lines to enhance soybean nutritional value without potential developmental tradeoffs.

TILLING by Sequencing: A Successful Approach to Identify Rare Alleles in Soybean Populations

Soybean seeds produce valuable protein that is a major component of livestock feed. However, soybean seeds also contain the anti-nutritional raffinose family oligosaccharides (RFOs) raffinose and stachyose, which are not digestible by non-ruminant animals. This requires the proportion of soybean meal in the feed to be limited, or risk affecting animal growth rate or overall health. While reducing RFOs in soybean seed has been a goal of soybean breeding, efforts are constrained by low genetic variability for carbohydrate traits and the difficulty in identifying these within the soybean germplasm. We used reverse genetics Targeting Induced Local Lesions in Genomes (TILLING)-by-sequencing approach to identify a damaging polymorphism that results in a missense mutation in a conserved region of the RAFFINOSE SYNTHASE3 gene. We demonstrate that this mutation, when combined as a double mutant with a previously characterized mutation in the RAFFINOSE SYNTHASE2 gene, eliminates nearly 90% of the RFOs in soybean seed as a proportion of the total seeds carbohydrates, and results in increased levels of sucrose. This represents a proof of concept for TILLING by sequencing in soybean.

Detection of novel allelic variations in soybean mutant population using Tilling by Sequencing

ABSTRACTOne of the most important tools in genetic improvement is mutagenesis, which is a useful tool to induce genetic and phenotypic variation for trait improvement and discovery of novel genes. JTN-5203 (MG V) mutant population was generated using an induced ethyl methane sulfonate (EMS) mutagenesis and was used for detection of induced mutations in FAD2-1A and FAD2-1B genes using reverse genetics approach. Optimum concentration of EMS was used to treat 15,000 bulk JTN-5203 seeds producing 1,820 M2 population. DNA was extracted, normalized, and pooled from these individuals. Specific primers were designed from FAD2-1A and FAD2-1B genes that are involved in the fatty acid biosynthesis pathway for further analysis using next-generation sequencing. High throughput mutation discovery through TILLING-by-Sequencing approach was used to detect novel allelic variations in this population. Several mutations and allelic variations with high impacts were detected for FAD2-1A and FAD2-1B. This includes GC to AT transition mutations in FAD2-1A (20%) and FAD2-1B (69%). Mutation density for this population is estimated to be about 1/136kb. Through mutagenesis and high-throughput sequencing technologies, novel alleles underlying the mutations observed in mutants with reduced polyunsaturated fatty acids will be identified, and these mutants can be further used in breeding soybean lines with improved fatty acid profile, thereby developing heart-healthy-soybeans.