Peanut is an important oil seed legume that is widely grown in all tropical and sub-tropical regions in the world (Bertioli et al., 2011). An important constraint to its production is contamination by aflatoxins, which are secondary metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus (Khlangwiset et al., 2011). The fungi occur ubiquitously in the soil and can colonize the seeds of a diverse number of crops such as peanut, maize, soybean, sorghum, cotton seed, cassava, chilies and tree nuts. At optimum conditions, typically temperatures between 24 and 35°C and humidity above 7%, the fungi can grow and produce aflatoxins on many commodities (Williams et al., 2004; Klich, 2007). A. flavus produces the aflatoxins B1 and B2 while A. parasiticus can also produce aflatoxins G1 and G2. Exposure to small amounts of these aflatoxins results in development of chronic aflatoxicosis while acute aflatoxicosis is the consequence of exposure to high concentrations of the toxins (Bhatnagar-Mathur et al., 2015). Both levels of exposure have serious consequences to health, since aflatoxin is the most potent naturally occurring carcinogen. It has been linked to development of liver cancer, suppression of the immune system, and nutritional interference, which in turn leads to severe weight loss and stunting especially in children (Williams et al., 2004). It is a serious problem especially in developing countries where high levels of exposure begin in utero and is associated with severe developmental challenges as well as growth impairment (Gong et al., 2002; Khlangwiset et al., 2011).

In many developing countries, there is high contamination of staple crops such as maize and peanut due to absence of regulations to control exposure (Wild and Gong, 2010). Combined with the challenges of frequent food shortages, consumption of highly aflatoxin-contaminated food is common (Guo et al., 2009). This was tragically illustrated in the 2004 aflatoxicosis outbreak in rural Kenya that resulted from consumption of maize with up to 1000 ppb of aflatoxin, leading to 125 deaths (Lewis et al., 2005). In developed countries, the law regulates the maximum aflatoxin contamination allowed. For instance in the US, only 0.5 ppb, 20 ppb, and up to 300 ppb respectively, is permissible in milk, food and feed products, respectively, while in the EU, limits as low as 4 ppb of total aflatoxins are allowed (Guo et al. 2009). In peanut, higher contamination rates result in destruction of nuts leading to losses that have been estimated at around $20 million annually in the southeastern US (Lamb and Sternitzke, 2001).

The health and economic consequences of aflatoxin contamination makes its prevention a key concern for the peanut industry (Holbrook et al., 2009). While it is possible to mitigate aflatoxin contamination by optimizing post-harvest cultural practices such as curing, drying and storage, a more desirable approach is the identification of genetic-based resistance to pre-harvest aflatoxin contamination (PAC). Ideally, resistant genotypes would be those that either mitigate fungal colonization, inhibit the formation of aflatoxin or exhibit resistance to abiotic stresses such as drought (Liang et al., 2006;Guo et al., 2008). Particularly for peanut, development of cultivars with resistance to PAC is desirable (Anderson et al., 1995; Holbrook et al., 2009; Wang et al., 2010). The geocarpic nature of the peanut plant, that is the habit of flowering above ground and production of fruit below the soil surface (Barker, 2005), makes the fruit particularly vulnerable to PAC.

The correlation of drought stress with development of PAC has been well established (Pettit et al., 1971; Hill et al., 1983; Wilson and Stansell, 1983; Holbrook et al., 2000a). The combination of water stress and exposure of developing peanut pods to high soil temperature, triggers the contamination of intact undamaged pods with aflatoxin prior to harvest. This is because A. flavus and A. parasiticus optimally grow at temperatures of 25 to 42°C, being able to thrive in soils with moisture potential as low as -35 MPa. Under these conditions, the fungi become extremely competitive and may become the dominant fungal species in the soil. Elevation of soil temperature occurs as solar radiation reaches the soil surface due to receding of the peanut canopy during severe drought (Blankenship et al., 1984; Cole et al., 1985; Payne, 1998). It is thus not surprising that in peanut, aflatoxin contamination becomes prevalent during prolonged exposure to heat and drought stress, with drought susceptible genotypes manifesting higher susceptibility to PAC (Holbrook et al., 2000a; Girdthai et al., 2010). As a result of the significance of aflatoxin, screening and breeding of cultivars with resistance especially to PAC is a key objective in peanut breeding (Holbrook et al., 2009; Wang et al., 2010).

Several studies have attempted to address the challenge of aflatoxin contamination, with availability of resistant genotypes being reported (Mixon and Rogers, 1973; Mehan et al., 1981; Mixon, 1986). These resistant genotypes were identified through in vitro seed colonization by A. flavus (IVSCAF) lab assays. However, when tested in field studies under drought conditions, these genotypes have not demonstrated clear genetic resistance to aflatoxin contamination (Blankenship et al., 1985; Anderson et al., 1995). Nonetheless, consistent with the association between drought stress and PAC, other studies have demonstrated that more drought tolerant genotypes accumulate less aflatoxin than drought susceptible genotypes. This was noted for example by Holbrook et al. (2000a) in a study that also noted positive correlations between PAC and leaf temperature measurements as well as visual stress ratings. Due to their cost effectiveness and ease of measurement, the latter two were considered to be potential proxies for selection of PAC resistance.

Due to the importance of the high oleic trait in peanut, studies have been done to determine whether differential accumulation of aflatoxin occurs with high oleic compared to high linoleic peanut. High oleic lines were shown to have high post-harvest growth of Aspergillus and increased aflatoxin contamination (Xue et al., 2003). However, a previous field-based study of high oleic peanut subjected to drought stress and aflatoxin inoculation did not show any differences in PAC as a result of fatty acid differences (Holbrook et al., 2000b). In a quest to find out if genetic resistance to other fungi that infect peanut can confer protection against Aspergillus, Holbrook et al. (1997) tested genotypes that are resistant to late leaf spot and white mold. Unfortunately, they did not observe reduced PAC in any of the genotypes tested, thus invalidating resistance to other fungi as an indirect selection method for PAC.

Recent studies of PAC in peanut seem to suggest the key to genetic resistance is not so much the prevention of fungal invasion but the diminution of aflatoxin production. For instance, using automated rainout shelters to induce aflatoxin contamination and RNA sequencing of susceptible genotypes, Clevenger et al. (2016) demonstrated that the permissive state for PAC occurs as a result of alteration of abscisic acid biosynthesis and signaling by the ABR1 gene. On the other hand, Korani et al. (2017) observed that under both field pre-harvest inoculation of A. flavus under drought stress and post-harvest inoculation, the drought tolerant genotype ICG 1471, was colonized with the fungus comparably with the susceptible genotypes, however, it accumulated less aflatoxin than the susceptible genotypes suggesting an underlying genetic mechanism that restricted aflatoxin accumulation.

In this study, we used a genomic selection (GS) strategy to screen two populations subjected to terminal drought stress for aflatoxin resistance. This took advantage of the availability of high density SNP chips for peanut (Clevenger et al., 2017; Clevenger, et al., 2018) and phenotype data from previous years that were used to derive prediction models for breeding values used for selection. The use of GS was first reported in cattle (Meuwissen et al., 2001), and has subsequently been used in rice (Spindel et al., 2015), wheat (Poland et al., 2012; Rutkoski et al., 2015; Battenfield et al., 2016), maize (Bernardo and Yu, 2007; Albrecht et al., 2011; Technow et al., 2013) and soybean (Zhang et al., 2016) breeding. The potential utility of GS in studying PAC resistance lies in the fact that it does not capitalize on detection of few large effect QTLs. Rather, it uses low cost genome spanning markers to detect even small effect variations that may not be captured by other QTL detection strategies (Heffner et al., 2009; Jannink et al., 2010). For studying PAC in peanut, this is useful, given the dearth of genetic variation for the trait (Anderson et al., 1995; Nigam et al., 2009). In addition to direct measurements of aflatoxin contamination, visual as well as high-throughput ratings were taken as easier to evaluate correlates for aflatoxin contamination.

Pre-harvest aflatoxin contamination is a serious constraint in peanut production as it renders the commodity unacceptable for trade and unfit for consumption as food or feed (Waliyar et al., 2016). While management practices are integral to amelioration of this problem, identification of genetic sources of resistance is desirable (Holbrook et al., 2009). This study attempted to take advantage of the numerous genomic resources now available for peanut to apply the novel method of GS in selecting for PAC tolerance. Genomic selection has been implemented with varying levels of success in studying traits with various levels of complexity in several species. For instance, prediction accuracies ranging from 0.16 to 0.34 for dry weight and 0.52 to 0.56 for days to heading have been reported in perennial ryegrass (Faville et al., 2016). In wheat, prediction accuracies of 0.35 for Fusarium head blight resistance, 0.62 for flour yield (Hoffstetter et al., 2016) and 0.28 to 0.45 for grain yield (Poland et al., 2012) have been reported. In maize, prediction accuracies of 0.28 to 0.49 for grain yield and 0.35 to 0.6 for plant height have been reported (Zhang et al., 2015), while in sugarcane, values of 0.22 to 0.45 for commercially extractable sucrose have been reported (Deomano et al., 2020). In peanut, cross validation analysis with multiple models have yielded prediction accuracies of 0.4 to 0.6 for pods per plant and shelling percentage, and above 0.6 for high heritability traits such as days to 50% flowering, days to maturity, 100 seed weight, oleic acid, rust resistance and late leaf spot (Pandey et al., 2020). Similarly, cross validation analysis of sting nematode resistance in peanut yielded prediction accuracies of 0.18 to 0.53 (Ravelombola et al., 2022).

In this study we began by implementing cross validation analysis on the Florida-07 X GP-NC WS 16 population. This population was comprised of 383 RILs that were developed to introduce TSWV as well as early and late leaf spot resistance to a cultivated background (Holbrook et al., 2013). Prediction accuracies ranged from 0.25 for TSWV to 0.79 for single seed weight (Table 1).

The tendency of traits with high heritability yielding higher correlation values was observed, which is in line with trends observed in other studies (Lin et al., 2014; Pandey et al., 2020). This was corroborated by available test data for ELS, single seed weight and single pod weight. For these traits, the GEBVs were correlated with the test data (Table 1). ELS which had moderate heritability had the lowest correlation between GEBVs and test data. Single seed and pod weights had high heritability. Correspondingly, their prediction accuracies were high and the correlations between GEBVs and test data were also high. The trend of prediction accuracy increasing with higher heritability indicated that GS was effective for selection of traits with a clear genetic basis. However, such traits are also easily amenable to conventional marker-assisted selection precluding the necessity of using GS, which would be more suitable for more complex, hard to phenotype traits such as aflatoxin.

The Tifrunner X C76-16 and Florida-07 X C76-16 populations were used to evaluate the possibility of using GS in breeding for the more challenging trait of PAC resistance. These populations have elite runner backgrounds (Tifrunner for Tifrunner X C76-16 and Florida-07 for Florida-07 X C76-16) with the un-adapted C76-16 used as a source of variability for drought tolerance (Holbrook et al., 2013).

Genotyping of the populations resulted in a total of 2204 and 1781 polymorphic SNPs for Tifrunner X C76-16 and Florida-07 X C76-16 populations, respectively (Table 2). The SNPs were evenly distributed in the chromosomes. For Tifrunner X C76-16, the percentage of polymorphic SNPs in each chromosome relative to total polymorphic SNPs ranged from 3.1% (A10) to 8.5% (B9) while for Florida-07 X C76-16 the range was 1.9% (A8) to 8.4% (B9) (Table 2). The genotype data were used for cross validation analysis. The prediction accuracy for PAC was 0.24 and 0.23 for Tifrunner X C76-16 and Florida-07 X C76-16, respectively. These low values corresponded to the low heritability scores of 0.31 and 0.1 for the two populations respectively (Table 3). Despite the low scores, the GEBVs were used to rank the populations, with the intention of testing the best and worst performers. Unfortunately, there was limited seed availability which impeded selection solely on the basis of the computed GEBVs. Twenty lines were selected for planting with a majority (15) being derived from the Tifrunner X C76-16 population. There were sufficient quantities of seed for 13 of the lines to be planted in two rainout shelters while the rest were planted in only one shelter (Table 4).

Preliminary exploration of data collected at the end of the season showed that all data were normally distributed. However, there were no significant differences between the lines for aflatoxin in both shelters and for visual drought ratings in shelter A (Table 5). In addition, spatial trends were observed, with most traits having higher or lower scores in plots along the edges of the shelters (Figure 1). This necessitated the use of models that accounted for spatial trends in the data for downstream analysis.

Rankings of aflatoxin content were inconsistent between the shelters. Similarly, the performance of the lines differed from the GEBVs, which were the basis of selection (Figure 2). In both shelters, differences were apparent both in rank and magnitude between the aflatoxin GEBVs and the assayed aflatoxin content values. These differences were more pronounced in shelter A. Correlation analysis showed low positive correlation (0.32) between the GEBVs and shelter B aflatoxin content. However, this was not the case for shelter A where the correlation was hardly discernible (-0.15) (Figure 3). These results serve to underscore the challenge of studying PAC which is extremely variable even with setups designed to induce heat and drought stress over extended periods as is necessary for consistent aflatoxin contamination (Holbrook et al., 1994, 2009).

Ideally, a difficult to phenotype trait such as PAC resistance would be preferable for implementing GS, since it uses whole genome spanning markers developed at low cost to detect small effect genetic variations that influence the trait (Heffner et al., 2009; Jannink et al., 2010). Despite the fair distribution of SNPs along the chromosomes of the two populations (Table 2), the parents of these populations have been shown to have generally low polymorphism (Chu et al., 2018). In addition, the process of advancing the population during RIL development may have favored selection of plants with minimal linkage drag from the common parent (C76-16), thus imposing population structure. Visual inspection of principal coordinate analysis plots of the SNP data revealed the likely presence of population structure in both populations (Figure 4). However, factoring this in the computation of GEBVs did not improve the analysis. Moreover, selection was done under irrigated conditions with no aflatoxin pressure. It is not known if genetic components from C76-16 with good aflatoxin or drought resistance made it to the RILs. These factors likely compromised the genetic architecture of PAC resistance, which of itself is a low heritability trait. Hence, in this study, GS does not confer clear advantages over conventional selection for PAC resistance.

In other studies, low visual stress ratings and leaf temperature - which are less variable and cheaper to measure - have been associated with reduced PAC in peanuts (Holbrook et al., 2000a; Girdthai et al., 2010). In this study, it was not possible to make such an association since the correlations between visual drought ratings and PAC were low (0.23 and 0.16 for shelter A and B, respectively). However, strong inverse correlations were observed between GNDVI and visual drought ratings (~ -0.7 in both shelters). In shelter B, GNDVI had comparable correlation with aflatoxin content as the GEBVs (Figure 5). Even though GNDVI is a high throughput phenotyping method that can substitute manual, subjective and labor-intensive drought rating methods, its predictive ability for aflatoxin resistance cannot be ascertained since the results did not replicate between the shelters. It is worth noting that GNDVI was the trait with the strongest correlation with yield in both shelters. Given that both traits are statistically significant, it can be deduced that GNDVI can be a good predictor of yield under drought conditions.

To the best of our knowledge, this is the first report of deployment of GS to tackle the challenge of breeding for PAC resistance in peanut. We also report the first use of GEBVs for selection in peanut. The use of genome spanning SNP markers ideally facilitates the detection of genetic variations that individually have a small effect on phenotype and are difficult to capture by other methods. Consequently, the method was demonstrated to be effective for traits with high heritability. Despite its potential usefulness for PAC resistance studies, the low heritability of the trait in the two populations studied resulted in weak prediction accuracies, suggesting that the challenge of genomic aided breeding for PAC resistance still remains. In addition, despite using rainout shelters to impose terminal drought stress, extreme variability of the trait was still observed. Lack of clear association of both visual drought ratings and GNDVI with PAC hindered the use of these strategies as better correlates for the trait.