Soybean is one of the most important oilseeds and affordable protein sources worldwide. Currently, the U.S. is the second-largest soybean producer globally, with soybean being its top revenue-generating crop after corn [1]. In line with the national trend, in 2022, soybean was planted on over 400,000 acres in South Carolina, acreage more than any other crop grown in the state, and produced a revenue of over $193.3 million [2]. High temperature has been identified as a primary environmental factor limiting soybean yield in the U.S., and the southeast U.S. is not an exception to this trend [3]. An increase of 1°C during the growing season was predicted to cause a 17% loss in yield [4,5]. Heat stress reportedly causes soybean yield suppression up to 6% daily under rainfed conditions if the growing season temperature is over 30°C [6]. With soybean being mostly a rainfed crop and climate change-associated high temperatures being a regular occurrence during its growing season, it is expected that heat stress will become a serious threat to soybean production worldwide and in the southern U.S. [3,6]. Additionally, with the early soybean production system (ESPS) adaptation in the mid-southern U.S., reduced germination has become a significant production problem. Under the ESPS, early-planted, short-season cultivars generally mature during the humid and warm periods between mid-August to mid-September [7]. During this period, the consistently elevated temperatures, which coincide with seed maturation, impair seeds physiologically in their ability to germinate and/or emerge successfully, making seeds unsuitable for soybean production [7]. The most plausible solution to these problems is to develop soybean genotypes with heat tolerance, which is constrained by the complex genetics of this trait and the unavailability of molecular markers to track this trait through generations in a breeding program. The current research project is a step forward in this direction, as it supplements the efforts put forth in an allied project [recently funded by USDA NIFA (2022-67013-36173)] by focusing on the development of DNA markers for heat-stress tolerance in soybean.

The major challenge that soybean breeders face is the lack of high-throughput and affordable screening methods for heat tolerance, notably the unavailability of suitable molecular markers. One reason is the quantitative nature of the heat tolerance, which makes it difficult to identify single major genes contributing to the trait. Our earlier research on soybean has demonstrated that a decrease in the level of lipids containing 18:3 acyl chains (linolenic acid) under heat stress in the tolerant genotype (DS25-1) is a likely consequence of the reduced activity of the FAD3A and FAD3B genes and contributes to its heat-tolerance [8]. These results corroborated with an earlier finding where the correspondence between heat-induced suppression of the FAD3A, FAD3B, and FAD3C expression levels and seed linolenic acid content was observed [9]. In line with these observations, transgenic silencing of the endoplasmic reticulum-localized fatty acid desaturase gene, FAD3, in soybean [3] and tomato [10], and the chloroplast-localized fatty acid desaturase gene, FAD7 in tomato [11] enhanced tolerance of these plants to heat stress. Additionally, the biochemical analyses of seed proteins isolated from DS25-1 (heat tolerant) and DT97-4290 (heat-sensitive) revealed that lipoxygenase (LOX), the lipid catabolism enzyme, the ß-subunit of ß-conglycinin (ßCG), sucrose binding protein (SBP), and Bowman-Birk protease inhibitor (BBI), differentially accumulate between these genotypes under heat stress [12]. These earlier studies provide convincing evidence that heat-induced changes in the lipid biosynthesis and metabolism genes correspond with heat-induced changes in cell membrane composition that confer adaptability to heat stress in soybean and bestow heat tolerance.
In sum, these results are promising as they imply that specific changes in lipid metabolism and seed proteome, contributing to heat stress tolerance, are controlled by a few genes. Moreover, the screening for the heat-induced changes in expression patterns or protein accumulation of these genes will allow the identification of genotypes with heat tolerance.

The purpose of our USDA project is to find associations between random genomic SNPs, i.e., markers distributed throughout the soybean genome, and heat-induced lipid metabolic changes and physiological traits. In contrast, this project focuses on identifying sequence polymorphism (substitutions, insertions, and deletions) within the candidate genes (CGs), i.e., genes identified to be involved in lipid metabolism (FAD genes) or differently expressed under heat stress (LOX, ßCG, SBP, BBI, MIPS1, MRP-L, and MRP–N) and use them in association analysis. These markers, once developed, will add significantly to the repertoire of molecular markers to be used and validated under the USDA project. We propose to use the parental genotypes DS25-1 and DT97-4290 of a recombinant inbred line (RIL) population for the detection of sequence polymorphisms in the CGs and RIL population to study the association between the heat-induced changes in expression patterns of the soybean FAD, MIPS1, MRP-L, and MRP–N genes or accumulation of LOX, ßCG, SBP, and BBI proteins, sequence variation(s) in CGs, and heat stress tolerance. Our preliminary and earlier studies show that the parental genotypes differ significantly in lipid and protein traits and germinability upon exposure to heat stress. Once identified, these markers will be used to breed heat tolerance in soybean genotypes, and the RILs will be used to transfer and stack this trait to the adapted germplasm. Such heat-tolerant soybean genotypes are critically needed to sustain soybean production in changing climatic conditions.

The two major storage constituents of soybean seed are oil and proteins. Lipids are the major components of biological membranes, which act as the interface between the cell and the environment. The structure and function of cells depend on the fluidity and stability of membranes, which are determined by lipid composition and unsaturation levels. Cis double bonds commonly present in most plant cell membrane fatty acyl chains introduce bends in the chains and reduce the degree of compact packing of adjacent lipid molecules. The compactness of packing is also reduced by heating. Decreasing the number of double bonds at high temperatures can be an adaptive mechanism in plants to maintain the optimal lipid packing, fluidity, and integrity of membranes, which is critical for maintaining the normal functioning of the photosynthetic machinery during stress [3,13]. Similarly, proteins serve many structural and metabolic functions, and their accumulation at the right time, space, and quantity are vital for maintaining proper structure and function. An earlier study demonstrated that heat stress severely impacts the accumulation of LOX, ßCG, SBP, and BBI in the susceptible genotype. In contrast, their accumulation in the tolerant genotype remains unaffected [12]. Further, reduced seed phytic acid accumulation enhanced susceptibility to heat stress and reduced germination [13]. Therefore, we hypothesize that decreasing lipid unsaturation levels and maintaining an optimal amount of vital proteins, and phytic acid content are associated with plants' heat-stress tolerance. We further propose that this decrease in lipid unsaturation is a consequence of reduced FAD activity, the unaltered protein level is an outcome of persistent LOX, CG, SBP, and BBI expression, and the amount of phytic acid in seeds is associated with the MIPS1, MRP-L, and MRP–N gene expression. All are associated with the sequence variations in these genes present between heat-tolerant and heat-susceptible genotypes. Earlier research in soybeans and other crops supported our hypothesis that the decline in the level of lipid unsaturation by decreasing the polyunsaturated fatty acids, such as linolenic acid, and increasing the less unsaturated fatty acids, such as linoleic, oleic, and palmitoleic acids, as well as saturated fatty acids such as palmitic and stearic acids and consistent accumulation of vital proteins and phytic acid are associated with heat tolerance [8,9,12]. Thus, in this proposal, we laid out the following objectives to validate the association between the heat-induced reduction in the expression levels of the soybean FAD genes or persistent expression of genes encoding essential proteins (LOX, ßCG, SBP, BBI, MIPS1, MRP-L, and MRP–N), heat stress tolerance, and sequence variation in these genes, and use this knowledge to breed for heat tolerance. 1) Determine the effect of heat stress on seed characteristics and germinability of a RIL population derived from a heat-tolerant and a heat-susceptible soybean genotype. The purpose of this experiment is to identify RILs showing extreme phenotypes upon exposure to heat stress. 2) Evaluation of the soybean genes encoding FADs, LOXs, ßCG, SBP, BBI, MIPS1, MRP-L, and MRP–N for substitutions, insertions, and deletions in a heat-tolerant and a heat-susceptible soybean genotype and RILs that exhibit extreme phenotypes upon exposure to heat stress via DNA sequencing. 3) Determination of association between the DNA sequence variations in candidate gene-base markers and the heat-tolerance traits using RILs that exhibit extreme phenotypes under heat stress (ten RILs from each end of the population phenotypic distribution) via multiple regression analysis.

Key Performance Indicators:
1) Development of DNA markers for heat tolerance, facilitating the transfer of this trait to other relevant genotypes. 2) Identification of genotypes from the RIL population, derived from a cross between DS25-1 (heat tolerant) and DT97-4290 (heat susceptible) for release as germplasm to breeding heat tolerance in other adapted lines. 3) Communication of results to the board members via the quarterly reports and to the breeders and producers via scientific publications and presentations.

The heat-tolerance-associated molecular markers and heat-tolerant RILs identified during this study will serve as the raw material for breeding soybean for heat tolerance. Such genotypes are critically needed to sustain soybean production in the face of the predicted rise in the global temperature and to meet the future soybean demand. By identifying markers associated with heat tolerance in soybean, breeders can then use these markers to produce a more adapted soybean for SC. This would give growers in SC the ability to sustainably produce soybean crops under heat stress, which is of common occurrence in this state.