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

Naoufal Lakhssassi,Shiming Liu,Leandro G Neves,Mallory A Cullen,Dounya Knizia,Abdelhalim El Baze,Mohamed G Embaby,Oumaima Chetto,Khalid Meksem,Zhou Zhou,Oussama Badad

doi:10.3390/ijms22084219

Abstract

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.

Highlights

Soybean [Glycine max (L.) Merr.] is the world’s largest oilseed crop and provides about 53% of vegetable oil in the U.S [1]
Gene editing using CRISPR has emerged as a promising technology for functional gene analysis in soybeans; it is still very limited in its use and requires highly trained laboratories and focus only on few genes
There is a strong need for new technologies combining high throughput screening methods while keeping lower cost for gene functional characterization

Summary

Introduction

Soybean [Glycine max (L.) Merr.] is the world’s largest oilseed crop and provides about 53% of vegetable oil in the U.S [1]. Soybean oil has a wide range of utilization in human consumption, animal feeding, and industrial applications. Modification of the five major fatty acid content in soybean oil draws much attention, as well as the production of novel fatty acids for nutritional enhancement [2]. The soybean research community has been focused on the metabolic engineering of fatty acid biosynthesis pathways to genetically improve soybean oil composition traits using different approaches [3]. Fatty acid desaturases are a large group of important enzymes that control saturated/unsaturated fatty acid ratio in soybean seed. There are three major fatty acid desaturase gene families in soybeans, which are localized at either plastid or endoplasmic reticulum (ER)

Methods

Results

Discussion

Conclusion