FAD: Fatty acid desaturase, PCR: Polymerase chain reaction, QTL: Quantitative trait loci, SSR: Simple sequence repeats.

Soybean [Glycine max (L.) Merr.] is the world’s leading oilseed crop valued for food and oil extraction, containing 40% protein, 20% oil, 35% carbohydrates and 5% ash (Lui, 1997; Alam et al., 2023). Soybean oil (18-21%) mainly comprises palmitic (10-12%), stearic (4%), oleic (20-23%), linoleic (53-55%) and linolenic (8-11%) acids (Wilson, 2004; Rathod et al., 2021). High oleic acid improves oxidative stability, extends shelf life, reduces hydrogenation and benefits health, while high polyunsaturated fatty acids reduce stability and favor trans-fat formation (Hu and Willett, 2002; Jerish et al., 2025).
       
Oil biosynthesis starts in chloroplasts with stearic acid, followed by desaturation in cytoplasm and endoplasmic reticulum through ∆⁹ desaturase, FAD2 (oleate to linoleate) and FAD3 (linoleate to linolenate) (Shanklin and Cahoon, 1998; Ohlrogge and Browse, 1995; Buchanan et al., 2015). Soybean has multiple FAD2 gene copies owing to the repeated rounds of genome duplications (Okuley et al., 1994). Seed-specific isozymes GmFAD2-1A/B regulate oleic acid during seed development, while GmFAD2-1C and GmFAD2-2 variants are expressed in cool climates and vegetative tissues (Schlueter et al., 2007).
       
Improving oil quality requires lowering polyunsaturated and enhancing monounsaturated fatty acids (Liu and White, 1992). Linolenic acid imparts off-flavors, while oleic acid reduces hydrogenation and trans-fat risk (Willett and Ascherio, 1994). Genetic modifications, especially FAD2-1a/b mutations, significantly increase oleic acid (Clemente and Cahoon, 2009; Richardson, 2016). While both the FAD2-1A and FAD2-1B mutant alleles, together are responsible for the elevated oleic acid levels (Kulkarni et al., 2018). Mutation and molecular breeding approaches, allow large-scale screening (Patil et al., 2010) whereas Near-Infrared Transmittance Spectroscopy facilities are accessible to limited researchers only and it is not perfect, as it does not guarantee the consistency (Gao et al., 2024) while, conventional Gas-Liquid Chromatography is seed destructive method.
       
NRC 147 (IISR, Indore) with 42% oleic acid (Singh, 2024), was crossed with Phule Agrani (KDS 344), Phule Sangam (KDS 726), Phule Kimaya (KDS 753) and genotypes KDS 869 and KDS 980 (20% oleic acid). Oleic acid levels are strongly affected by environmental conditions such as temperature, rainfall and photothermal interactions (Pham et al., 2012), making phenotype-based selection unreliable in early generations. Marker-assisted selection enables rapid, cost-effective identification of desirable alleles. Thus, F₂ and F₃ segregants were used to fix high-oleate traits early and accelerate breeding progress. Instead of conventional gas-liquid chromatography is destructive, marker-assisted selection offers a non-destructive and convenient alternative. Hence, this study aimed to screen soybean segregants for oleic acid using molecular markers.

The F₂/F₃ seeds of five different crosses of soybean were used with NRC147 being the common donor parent for high oleate content and the details are listed in Table 1.


 
Molecular marker-based analysis of oleic acid trait in soybean
 
Healthy seeds of crosses involving six parents viz NRC 147 (donor) possess about 42% oleic acid content (Singh, 2024), Phule Agrani (KDS 344), Phule Sangam (KDS 726), Phule Kimaya (KDS 753), KDS 869 and KDS 980 were grown for DNA extraction along with 110 segregants derived from their crosses. Genomic DNA was isolated from tender leaves using a modified CTAB method (Doyle and Doyle, 1990) and the final DNA concentration was adjusted to 30 ng/µl for PCR amplification.
 
Designing of primers for simultaneous amplification of both FAD2-1A/FAD2-1B gene
 
1. Full-length nucleotide sequences of FAD2-1A (AB188250.1) and FAD2-1B (AB188251.1) were retrieved using “Somewhat Similar Nucleotide BLAST”, selecting hits with >97% query coverage and >99% identity. InDel/SNPs in the intron region were identified and primers were designed to be polymorphic between the two genes while conserved at annealing sites.
2. Cluster Alignment with Weights (ClustalW) was used for Multiple Sequence Alignment (MSA) selecting conserved flanking regions around insertion-deletion polymorphisms.
3. Primer BLAST was used for designing the FAD2-1A gene-specific primers.
4. In silico screening of primer pairs was carried out to confirm specificity, expected product size and to check possible amplification of FAD2-1B.
 
FAD2-1 isoform specific and SSR markers used to determine oleic acid gene in soybean
 
SSR markers linked to unsaturated fatty acid composition (Bachlava et al., 2009; Priolli et al., 2015) were used to analyze segregants. Isoform-specific primers for FAD2-1A and FAD2-1B (Bachlava et al., 2008) were employed to amplify the coding regions of respective isoforms. Primer sequences as mentioned in Table 2 were custom-synthesized by BioServe Biotechnologies (India) Pvt. Ltd., supplied in lyophilized form and were diluted before use for PCR.


 
PCR amplification
 
PCR reactions were set up in 0.2 ml tubes with 30 ng template DNA, 1 U Taq DNA polymerase, 1X Taq buffer-B, 1.0 mM MgCl₂, 1 µM dNTP mix and 20 pmol of each primer. The amplification regime comprised of initial denaturation (95°C, 5 min), 30 cycles of denaturation (94°C, 1 min), annealing (41-54°C, depending on primer) and elongation (72°C, 90 s), followed by a final elongation (72°C, 10 min). Amplification was performed in an Eppendorf Thermal Cycler.
       
PCR products were resolved by agarose gel electrophoresis: 2.5% for SSR primers and 1.6% for FAD2-specific primers and run at 80 V for 1.5-2 h followed by gel image analysis.

The study evaluated 110 soybean segregants from five crosses: 40 F₂ segregants (30 from KDS 980 × NRC 147; 10 from KDS 344 × NRC 147) and 70 F₃ segregants (35 from KDS 869 × NRC 147; 30 from KDS 726 × NRC 147; 5 from KDS 753 × NRC 147). FAD2 gene-specific primers and SSR markers linked to seed oleic acid QTLs were used to assess functional (FAD2-1) and dysfunctional (fad2-1) alleles in individual segregants (Table 3).


       
FAD2-1A / FAD2-1B primers were designed during this study by comparing the FAD2-1A gene (accessions AY954300.1, ACUP0300731.1) with FAD2-1B gene (accession AB188251.1). Out of 2662 bases were studied in multiple sequence alignment, 1801 bases aligned with each other of which 1564 (86.84%) were identical (Fig 1). Out of 406 bases in intron region, only 312 base sequences showed similarity. Due to high variation in the intron region, this region was selected for designing primers specific to FAD2-1A/FAD2-1B isoforms for their simultaneous amplification.


       
A parental polymorphism study was conducted using ten primer pairs on six parental cultivars: NRC 147 (high oleate donor), Phule Agrani (KDS 344), Phule Sangam (KDS 726), Phule Kimaya (KDS 753) and genotypes under field trials KDS 869 and KDS 980. PCR amplification showed that seven primers (IFAD2-1A, IFAD2-1B, Sat_108, Satt294, Satt386, Satt002 and Satt001) were polymorphic (Table 3).
The detailed parental polymorphism results are as follows.
 
• IFAD2-1A
 
This isoform-specific primer (GenBank accessions AB188250; Benson et al., 2002) amplified 155 bp in KDS 980, 162 bp in KDS 869 and KDS 753, whereas 140 bp in KDS 726, KDS 344 and NRC 147 (Table 3). As it did not amplify a unique NRC 147-specific band and hence was not considered for individual plant analysis.
 
• IFAD2-1B
 
This isoform-specific marker (GenBank accessions AB188251; Benson et al., 2002) amplified dual 284 bp and 190 bp bands in low oleate parents, while 163 bp band in NRC 147 (Table 3). Although polymorphic and suitable for oleate trait analysis, due to its low annealing temperature (41°C) it was not considered further.

• Satt354
 
PCR amplification amplified 190 bp band in NRC 147, while it did not amplify in rest five parentsin repeated attempts (Table 3; Fig 2a-2h, 3a-3h). Thus, Satt354 behaved as a null allele in low-oleate parents and was selected as a candidate marker for individual plant studies. Both Satt354 and the GmFAD2-1Bgene isoforms are reported to be located on chromosome number 20.


 
• Sat108
 
Positioned on Chromosome 10(O), Sat_108 amplified product sizes from 207-234 bp: 207 bp (KDS 980), 213 bp (KDS 869, KDS 753), 220 bp (KDS 726), 226 bp (KDS 344) and 234 bp (NRC 147) (Table 3). The 234 bp allele specific to NRC 147 makes Sat_108 informative for individual plant studies of the oleate trait.
 
• Satt294

Positioned on Chromosome 4(C1) at the Seed oleic 1-g5 locus, Satt294 amplified 280 bp in NRC 147, 290 bp in KDS 344, KDS 726 and KDS 753 and 296 bp in KDS 869 and KDS 980 (Table 3 and Fig3a-3h). The distinct 280 bp allele of NRC 147 makes Satt294 an informative marker for individual plant studies of the oleate trait.


 
• Satt386
 
Linked to Seed oleic 1-g11 QTL on Chromosome 17(D2), Satt386 amplified 200 bp in NRC 147 and 190 bp in the other five parents (Table 3; Fig2a-2h). This NRC 147-specific polymorphism makes Satt386 suitable for individual plant analysis of the oleate trait.
 
• Satt002
 
Associated with QTL Seed oleic 1-g10 on Chromosome 17 (D2), Satt0025 parents amplified a 127 bp band, while KDS 869 produced a 140 bp band (Table 3). It was non-informative for studies of the oleate trait.

• Satt487
 
Associated with QTL Seed oleic 1-g33 on Chromosome 10 (O), Satt487 produced a monomorphic 200 bp band across all parents (Table 3).
 
• Satt001
 
This marker is linked to Seed oleic 1-g26 QTL on Chromosome 9 (K). It amplified product sizes of 215 bp and 207 bp, in NRC 147 and KDS respectively, while rest four parents amplified a 203 bp PCR product (Table 3). Thus, Satt001 exhibited polymorphism among the parents and is informative for individual segregant analysis.
 
• FAD2-1A/FAD2-1BSpecific
 
The FAD2-1A/FAD2-1B primers designed in this study specifically amplified both FAD2-1A and FAD2-1B genes, producing expected twin PCR products of 401 bp and 378 bp, respectively. Amplification in all six parents was monomorphic, with the twin products corresponding to the isoforms, distinguished by three InDel sites (7, 9 and 8 bp) in FAD2-1B. These primers allow tracking of fad mutant alleles. Earlier studies have shown that Single mutants (FAD2-1 aaBB, 34.4%; FAD2-1 AAbb, 26.2%) showed higher oleic acid than the wild type (FAD2-1 AABB, 21.3%), while the double mutant (FAD2-1aabb) had the highest oleic content (77.1-81.8%) (Richardson, 2016). Molecular analysis revealed that variations in FAD2-1A affected conserved histidine residues required for enzymatic activity, whereas FAD2-1B mutations introduced a premature stop codon, terminating translation (Lee et al., 2019).
 
PCR amplification for screening soybean segregants
 
From parental polymorphism analysis, 4 informative primers viz Satt354, Satt001, Satt294 and Satt386capable of distinguishing NRC 147 from the rest five parents, with high annealing temperature (53°C), were used to screen individual plants from five crosses. Satt354 amplified only NRC 147 DNA, while multiplex PCR with Satt294 and Satt386 enabled simultaneous detection of multiple alleles in segregating plants for oleic acid content. Molecular markers linked to QTLs revealed genomic regions controlling soybean oil traits (Priolli et al., 2015). In their study over two years, 33 SSR loci were associated with oleate, followed by linoleate (26), palmitate (24) and linoleate (14). 8 loci with specific alleles contributed to high-performing soybeans, indicating the usefulness of these markers for rapid improvement of oil quality.
 
Satt386 and Satt354
 
Satt386 and Satt354 were used to screen 110 individual segregants (Fig 2a-2h). Satt386 amplified a 190 bp band from NRC 147 and one plant showed dual bands (190 bp from NRC 147 and 200 bp from KDS 344). Among 30 F₂ segregants of KDS 980 × NRC 147, 12 amplified the 190 bp band, while 2 of 10 F₂ plants of KDS 344 × NRC 147 also amplified the 190 bp band. In F₃ segregants, 13 out of 30 (KDS 869 × NRC 147), 10 out of 30 (KDS 726 × NRC 147) and 1 out of 5 (KDS 753 × NRC 147) amplified the 190 bp band. As both Satt386 and Satt354 amplified this 190 bp fragment in NRC 147 and derived segregants, detailed Satt354 results were further analyzed using Satt294 and Satt354 multiplex PCR to distinguish additional alleles. Similar studies in sunflower revealed that high-oleic individuals (62.49-93.82%) could be effectively identified using SSR markers like F4-R1, whereas low-oleic plants (15.24-31.28%) did not amplify the marker (Dimitrijević et al., 2017).
 
Satt294 and Satt354
 
Two SSR primers, Satt294 and Satt354, were used to study individual segregants (Fig 3a-3h). With Satt294, 280 bp bands corresponding to NRC 147 were amplified in 11/30 (KDS 980 × NRC 147), 3/10 (KDS 344 × NRC 147), 16/35 (KDS 869 × NRC 147), 11/30 (KDS 726 × NRC 147) and 1/5 (KDS 753 × NRC 147) segregants. Earlier, Monteros et al. (2008) identified six oleic acid QTLs using SSRs, enabling MAS for high-oleate breeding.
       
With Satt354, a 190 bp band was detected in 3/30 (KDS 980 × NRC 147), 10/10 (KDS 344 × NRC 147), 22/35 (KDS 869 × NRC 147), 11/30 (KDS 726 × NRC 147) and 3/5 (KDS 753 × NRC 147) segregants, while acting as a null allele in low oleate parents. This primer maps to Chromosome 20, where FAD2-1B gene resides. Bachlava et al., (2008) also mapped FAD2 isoforms to LGs O, I and L, linking them to oleate QTLs.
       
Overall, Satt386, Satt294 and Satt354 together identified 38, 42 and 39 segregants, respectively, for NRC 147 alleles. These are being advanced for further correlation study of marker pattern with oleic acid profile.

Out of the 10 molecular markers studied, Satt386, Satt294 and Satt354 were most informative for screening individual segregants to reveal 38 (F₂: 14, F3: 24); 42 (F₂: 14, F₃: 28) and 39 (F₂: 3, F₃: 36) segregants, respectively, with markers matching NRC 147, the higher oleate parent. Further, FAD2-1A/ FAD2-1B specific primers were developed, which differentially amplified 401 bp and 378 bp markers simultaneously for those two isoforms. The identified markers may potentially contribute to breeding favorable soybean oil characteristics.

We acknowledge the Mahatma Phule Krishi Vidyapeeth, Rahuri for providing funds to carry out the above research. We thank Officer Incharge, State Level Biotechnology Centre, M.P.K.V., Rahuri for allowing us to undertake this research work.
 
Authors’ contributions
 
Author wise contribution to the study’s conception and design (VPC), study of the work (AHG), material preparation (MPD), data collection (AHG), analysis (AHG and VPC) and manuscript (VPC, ONR and ARA). All authors have read and approved the final manuscript.
 
Compliance with ethical standards
 
Authors declare that they don’t have any conflict of interest and that there are no ethical issues.

Authors do not have any conflict of financial or non-financial interests to declare/disclose that are directly or indirectly related to the work submitted for publication.