**2. Breeding goals for improvements in the ω-3 fatty acid content of soybean seeds**

ALA (ω-3) in soybean oil is unstable and has undesirable flavors. Due to the presence of a double bond at the 12th carbon in the fatty acid hydrocarbon chain, the ALA is oxidized easily, which causes unwanted odor and off-flavors [29–31]. Ultimately, this reduces the functional quality of fry food or soy food items [31, 32]. Hence, ALA content is negatively associated with the stability and shelf life of soybean oil. To improve the shelf life, stability, desirable flavor, and palatability, soybean oil is chemically hydrogenated, which leads to the formation of *trans* fats. The *trans* fatty acids are linked to coronary heart diseases, and hence it is desirable to reduce the consumption of foods containing *trans* fats. Nearly 13 million Americans are reported to suffer from coronary heart disease, and over 500,000 die annually from causes related to coronary heart disease. For these reasons, the addition of *trans* fat labels to food nutrition labels directly under the line for saturated fat was started in 2006. This resulted in a change in the narrative of food industries to promote and explore substitutions for hydrogenated soybean oils. In the past few decades, soybean geneticists and breeders have shown that oil with reduced *trans*-fat content can be obtained by decreasing the content of ω-3, and ω-6 fatty acids through conventional and modern breeding approaches. Soybean oils derived from cultivars with reduced ω-3 fatty acid content are shown to increase stability, lower hydrogenation, and comprise reduced *trans*-fat levels [29, 33, 34]. This shifted the breeder's focus to develop soybeans with reduced ω-3 fatty acid content [35, 36]. In this section, the genetic basis of ALA (ω-3) content and breeding approaches fro altering ALA content are discussed.

#### **2.1 Genetic basis of PUFA in soybean**

The biosynthesis of PUFAs in soybean involves a variety of pathways, which are catalyzed by a complex series of desaturation and elongation steps [37]. Fatty acid desaturases introduce double bonds into the hydrocarbon chains of fatty acids to produce unsaturated fatty acids [38]. The delta-12 fatty acid desaturase-2 enzyme (*FAD2*) catalyzes the conversion of oleic acid to linoleic (ω-6) in the developing soybean seeds [39]. The microsomal ω-3 fatty acid desaturases (*FAD3*) catalyze the transformation of linoleic into ALA [36]. Thus, the genes coding for these fatty acid desaturases may act together to control the ALA content in soybean.

Two identical copies of FAD2 enzymes (FAD2-1, and FAD2-2) have been identified in the soybean. Five *FAD2* gene family members (two *FAD2-1* members: *FAD2-1A* and *FAD2-1B*, and three *FAD2-2* members: *FAD2-2A*, *FAD2-2B*, and *FAD2-2C*) are present in the soybean genome [39, 40]. Through syntenic, phylogenetic, and in silico analysis, Lakhssassi et al. [41] revealed two additional members of the *FAD2* gene family: *FAD2-2D* and *FAD2-2E*, positioned on chromosomes 9 and 15, respectively. Of these *FAD-2* genes, *FAD2-1A* is highly expressed in developing soybean seeds [41]. The chromosomal locations and gene model names of these genes are given in **Table 2**. Mutations in one or more of these genes have been utilized to alter the fatty acid content of the soybean seeds.

The genetic basis ω-3 fatty acid trait in soybean has been identified based on the experimental study of gene information from the model plant *Arabidopsis thaliana* (L.) Heynh. and screening of lower ω-3 fatty acid mutant soybean lines. Arabidopsis contains a single gene encoding a microsomal ω-3 fatty acid desaturase and two chloroplast targeted enzymes (FAD7 and FAD8). At least three independent loci (*fan, fan2*, and *fan3*), influencing the ALA content have been identified [42]. Genes underlying all the three loci have been identified as homologous genes of *FAD3* [43]. In soybeans, the level of ALA is controlled by three *FAD3* genes: *FAD3A* (*Glyma.14g194300* for Wm82.a2.v1 assembly)*, FAD3B* (*Glyma.02g227200*)*,* and *FAD3C* (*Glyma.18g062000)*. Of these, *FAD3A* has been reported to show consistent high expression in developing seed, and hence have the greatest effect in controlling the accumulation of ALA contents [42, 44]. These three loci have a greater effect on ALA concentration, as combining mutant alleles of these genes resulted in soybean oil having ~1% ALA [45–48].

The full-length genomic DNA sequences for *FAD3A, FAD3B, FAD3C*, and *FAD3D* genes were found to share 78 to 95% similarity, and have similar structure, and contain eight exons [49]. These eight exons of *FAD3* genes are highly conserved in soybean and correspond to the sizes of exons in Arabidopsis. However, significant variation is found among the introns of *FAD3* genes. Based on the structure and similarity, the four *FAD3* genes could be separated into two groups: *FAD3A*/*FAD3B*, and *FAD3C*/*FAD3D.*

## **2.2 Reducing ω-3 fatty acid content to avoid the need for chemical hydrogenation**

Initial breeding efforts were made by the USDA-ARS in 1952 to identify soybean germplasm with lower ω-3 content to replace chemical hydrogenation of soybean oil [50]. During that period, the cultivars with lower ω-3 levels were identified but


#### **Table 2.**

*Chromosomal location and gene model information of FAD2 and FAD3 genes in the soybean genome.*

#### *Breeding Strategy for Improvement of Omega-3 Fatty Acid through Conventional Breeding… DOI: http://dx.doi.org/10.5772/intechopen.95069*

no cultivar having <4% ω-3 were found. In 1981, USDA-ARS and North Carolina State University collaborated and developed the line N79-2245 having a reduced ω-3 content of 4.2% by recurrent selection approach [32]. The major cultivars/lines with low ω-3 content developed using conventional breeding, germplasm screening, mutation breeding, and recurrent selection [44, 51–53] are given in **Table 3**.

The natural accessions reported with the low ω-3 level in the USDA germplasm is known as PI 123440 and PI 361088B with allelic variant at *fan* locus. This mutation was reported as allelic or identical to the initial single recessive allele derived from the C1640 genotype [57, 61–63]. Burton et al. [71] used the PI 123440 as a parent source to develop a low ω- trait known as Soyola and Satelite.

Through the EMS and X-ray mutagenesis approach, several mutants were previously reported for the lower ω-3 fatty acid content ranging from <2.5% to 5.6% that are linked with the *Fan* loci such as C1640 (*fan*), A5 (*fan*), A23 (*fan2*), KL-8 (*fanx*), M-5 (*fan*), M-24 (*fanxa*) and RG10 (*fan-b*) [54, 58, 60, 64, 66, 67, 72]. Besides, mutants A16, A17, A29, MOLL, and LOLL with reduced ω-3 acid content showed allelic variation at *fan* loci [58, 64, 66, 67]. The RG10 line was developed from the mutagenesis of 4% ω-3 line C1640 [55, 60]. Several studies


#### **Table 3.**

*List of soybean mutants and germplasm lines with low levels of* α*-linolenic acid in the seed oil.*

used the RG10 line to develop the novel lines (*GmFAD3aabbcc*) with low ω-3 fatty acid content and also used for mapping and validating the quantitative trait loci (QTLs) and *FAD3* genes [45, 49, 73]. The EMS mutant line PE1690 with the reduced ω-3 fatty acid was reported to have a single base mutation in the *FAD3A* gene, resulting in the desaturase enzyme being nonfunctional [51]. Recently, Held et al. [69] identified a novel mutant allele of the *FAD3C* gene in a screen of a N-nitroso-N-methylurea (NMU)-mutagenized population. This allele resulted in 2 to 3% reduction in ω-3 FA levels.
