Innovative Development in High Stearic Acid Soybean Germplasm Breeding at University of Missouri Delta Center

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Innovative research on developing high stearic acid soybean germplasm is being conducted at the University of Missouri Delta Center. Challenges in breeding high stearic soybeans, such as sodium azide-induced deletions and mutant alleles in SACPDs, are being addressed. Promising lines identified from yield tests in 2021 show potential for germplasm release. Advanced techniques like machine-learning GWAS are aiding in identifying QTLs related to fatty acid biosynthesis. Genome sequencing is providing valuable insights into mutant alleles and structural variations in deletion regions.

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Development of Functional Ultra Development of Functional Ultra- -High Stearic Acid Soybean Germplasm Soybean Germplasm High Stearic Acid Grover Shannon, Pengyin Chen, Caio Canella, and Dongho Lee MSSB Project Meeting MSSB Project Meeting University of Missouri Delta Center Soybean Breeding August 2022

Soybean Breeding Delta Center Summary of the previous Stearic acid - Bakery products, shortenings, spreads, and margarines - Oxidative stability for industrial applications - Important parameter for oil quality High stearic soybean lines Challenges in breeding high stearic soybeans A6 - Sodium azide - 6Mbp deletion - SA: ~28% FAM94-41 - Natural - 1bp substitution - SA: ~9% - Low germination and negative agronomic traits SACPD-B TCJWB03-806-7-19 Known mutant alleles in SACPDs - Gamma ray - 1bp deletion - SA: ~7% - SACPD-B (Chromosome 2) - SACPD-C (Chromosome 14) SACPD-C 30-1947-1 - Fast Neutron - 2Mbp deletion - SA: ~9% Chr.2 Chr.14

Soybean Breeding Delta Center Summary of the previous Two mapping populations Out of 250 RILs, 60 lines entered preliminary yield test in 2020 and three lines were selected for advanced yield test in 2021. Advanced yield test in 2021 Yield (bu/ac) 50.8 47.9 47.8 XTa RRb Line Maturity Stearic acid (%) S19-19183 S19-19186 S19-19190 4.8 4.3 4.9 74.6 70.3 60.0 77.7 73.2 - 16.0 18.5 17.0 a the percentage of yield performance of lines compared to Xtend checks. Prolonged off-target dicamba exposure significantly affected the yield performance of non-Xtend soybean lines. b the percentage of yield performance of lines compared to Round-up Ready checks. Promising lines for germplasm release!

Soybean Breeding Delta Center Summary of the previous 6K SNP data GWAS result Smaller deletion size than A6 on SACPD-C regions on chromosome 14 Phenotypic variations showing that some lines have much higher stearic acid than 30-1947-1 Multiple mutant alleles or structural variations in deletion region? Markers around the significant marker (a) resulted from GWAS and SACPD-C (b) region and the SNP information and stearic acid (%) of selected RILs and high stearic mutants. Genome sequencing!!

Soybean Breeding Delta Center Progress in 2022 Machine-learning GWAS Enhanced statistical power to identify QTL with minor effect A total of nine SNP markers involved in fatty acid biosynthetic process chromosome 1, 3, 5, 13, 14, 16, and 18. were found on

Soybean Breeding Delta Center Progress in 2022 Sequencing strategy & field experiment 17 lines Whole genome sequencing Sample # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Group High Steric High Steric High Steric High Steric High Steric High Steric Low Stearic Low Stearic Low Stearic Low Stearic Low Stearic Low Stearic Pedigree Pedigree Historical line WT parents WT parents Line Stearic acid 2Mbp Deletion 21.1 20.5 19.2 14.2 16.6 16.5 3.5 3.4 3.3 3.2 3.1 3.1 9.1 7.7 22.0 6Mbp deletion 3.0 3.0 S19-19188 S19-19187 S19-19715 S19-19185 S19-19181 S19-19712 S19-18842 S19-18829 S19-20072 S19-20086 S19-20088 S19-20018 LLL-05-1 30-1947-1 A6 S13-10590C S14-9017R YES YES YES YES YES YES NO NO NO NO NO NO NO YES Whole genome sequencing has been conducted for dissecting structural variation in the deletion Analyzing structural variations is on processing (Time due is in November) Targeted sequencing Targeted sequencing will be conducted for confirming the presence of known mutant alleles Agronomic traits 2m-long single rows (100 seeds per row) were used for agronomic trait measurements (plant height, number of pods/nodes, 100-seed weight) NO NO

Soybean Breeding Delta Center New populations in 2022 progeny Stearic acid (%) Oleic acid (%) (average) Traits Population Pedigree # lines Range 20.2 - 20.9 21.0 - 34.3 20.0 - 23.6 20.8 - 25.1 20.1 - 33.4 21.7 - 21.8 20.0 - 27.4 21.3 6.3 - 14.9 7.0 - 17.2 8.8 - 18.4 11.7 7.4 8.4 Average 20.6 27.1 21.8 22.3 22.1 21.8 22.5 21.3 9.9 11.2 12.9 - - - UHS UHS UHS UHS UHS UHS UHS UHS HSHO HSHO HSHO HSHOLL HSHOLL HSHOLL CR20-132 CR20-133 PR20-038 PR20-039 PR20-040 PR20-042 PR20-043 PR20-044 CR20-131 PR20-042 PR20-044 PR20-038 PR20-042 PR20-043 S19-19710 x S16-11644C S19-19712 x S16-7922C S19-19710 x S17CR-172 S19-19181 x S16-5540R S19-19181 x S17CR-189 S19-19196 x S17CR-172 S19-19188 x S11-20337 S19-19188 x S17PR-482R S19-19715 x S17PR-481 S19-19196 x S17CR-172 S19-19188 x S17PR-482R S19-19710 x S17CR-172 S19-19196 x S17CR-172 S19-19188 x S11-20337 2 7 2 4 18 2 6 2 11 7 4 1 1 1 23.8 12.1 23.3 22.5 21.2 23.9 20.4 34.9 75.1 72.1 70.9 74.0 / 3.2 (18:3) 79.2 / 3.1 (18:3) 81.5 / 3.0 (18:3)

Soybean Breeding Delta Center What is next? Progeny Selection Agronomic traits Genome sequencing Germplasm Select the promising progeny lines for further multi- environmental yield tests Dissect structural variations in deletion and confirm the presence of known mutant alleles Germplasm release for research community and soybean producers Measure agronomic traits