Linkage Mapping and Genome-Wide Association Study Identified Two Peanut Late Leaf Spot Resistance Loci, PLLSR-1 and PLLSR-2, Using Nested Association Mapping.

Identification of candidate genes and molecular markers for late leaf spot (LLS) disease resistance in peanut (Arachis hypogaea) has been a focus of molecular breeding for the U.S. industry-funded peanut genome project. Efforts have been hindered by limited mapping resolution due to low levels of genetic recombination and marker density available in traditional biparental mapping populations. To address this, a multi-parental nested association mapping population has been genotyped with the peanut 58K single-nucleotide polymorphism (SNP) array and phenotyped for LLS severity in the field for 3 years. Joint linkage-based quantitative trait locus (QTL) mapping identified nine QTLs for LLS resistance with significant phenotypic variance explained up to 47.7%. A genome-wide association study identified 13 SNPs consistently associated with LLS resistance. Two genomic regions harboring the consistent QTLs and SNPs were identified from 1,336 to 1,520 kb (184 kb) on chromosome B02 and from 1,026.9 to 1,793.2 kb (767 kb) on chromosome B03, designated as peanut LLS resistance loci, PLLSR-1 and PLLSR-2, respectively. PLLSR-1 contains 10 nucleotide-binding site leucine-rich repeat disease resistance genes. A nucleotide-binding site leucine-rich repeat disease resistance gene, Arahy.VKVT6A, was also identified on homoeologous chromosome A02. PLLSR-2 contains five significant SNPs associated with five different genes encoding callose synthase, pollen defective in guidance protein, pentatricopeptide repeat, acyl-activating enzyme, and C2 GRAM domains-containing protein. This study highlights the power of multi-parent populations such as nested association mapping for genetic mapping and marker-trait association studies in peanuts. Validation of these two LLS resistance loci will be needed for marker-assisted breeding.

Mapping quantitative trait loci (QTLs) and estimating the epistasis controlling stem rot resistance in cultivated peanut (Arachis hypogaea).

KEY MESSAGE: A total of 33 additive stem rot QTLs were identified in peanut genome with nine of them consistently detected in multiple years or locations. And 12 pairs of epistatic QTLs were firstly reported for peanut stem rot disease. Stem rot in peanut (Arachis hypogaea) is caused by the Sclerotium rolfsii and can result in great economic loss during production. In this study, a recombinant inbred line population from the cross between NC 3033 (stem rot resistant) and Tifrunner (stem rot susceptible) that consists of 156 lines was genotyped by using 58 K peanut single nucleotide polymorphism (SNP) array and phenotyped for stem rot resistance at multiple locations and in multiple years. A linkage map consisting of 1451 SNPs and 73 simple sequence repeat (SSR) markers was constructed. A total of 33 additive quantitative trait loci (QTLs) for stem rot resistance were detected, and six of them with phenotypic variance explained of over 10% (qSR.A01-2, qSR.A01-5, qSR.A05/B05-1, qSR.A05/B05-2, qSR.A07/B07-1 and qSR.B05-1) can be consistently detected in multiple years or locations. Besides, 12 pairs of QTLs with epistatic (additive × additive) interaction were identified. An additive QTL qSR.A01-2 also with an epistatic effect interacted with a novel locus qSR.B07_1-1 to affect the percentage of asymptomatic plants in a row. A total of 193 candidate genes within 38 stem rot QTLs intervals were annotated with functions of biotic stress resistance such as chitinase, ethylene-responsive transcription factors and pathogenesis-related proteins. The identified stem rot resistance QTLs, candidate genes, along with the associated SNP markers in this study, will benefit peanut molecular breeding programs for improving stem rot resistance.

Genotypic effect of ahFAD2 on fatty acid profiles in six segregating peanut (Arachis hypogaea L) populations.

BACKGROUND: Fatty acid composition of oil extracted from peanut (Arachis hypogaea L.) seed is an important quality trait because it may affect the flavor and shelf life of resulting food products. In particular, a high ratio of oleic (C18:1) relative to linoleic (C18:2) fatty acid (O/L ≥ 10) results in a longer shelf life. Previous reports suggest that the high oleic (~80%) trait was controlled by recessive alleles of ahFAD2A and ahFAD2B, the former of which is thought to have a high frequency in US runner- and virginia-type cultivars. Functional mutations, G448A in ahFAD2A and 442insA in ahFAD2B eliminate or knock down desaturase activity and have been demonstrated to produce peanut oil with high O/L ratios. In order to employ marker assisted selection (MAS) to select a high oleic disease resistant peanut and to evaluate genotypic and phenotypic variation, crosses were made between high oleic (~80%) and normal oleic (~50%) peanuts to produce segregating populations. RESULTS: A total of 539 F2 progenies were randomly selected to empirically determine each ahFAD2 genotype and the resulting fatty acid composition. Five of the six crosses segregated for the high oleic trait in a digenic fashion. The remaining cross was consistent with monogenic segregation because both parental genotypes were fixed for the ahFAD2A mutation. Segregation distortion was significant in ahFAD2A in one cross; however, the remaining crosses showed no distortion. Quantitative analyses revealed that dominance was incomplete for the wild type allele of ahFAD2, and both loci showed significant additive effects. Oleic and linoleic acid displayed five unique phenotypes, based on the number of ahFAD2 mutant alleles. Further, the ahFAD2 loci did exhibit pleiotropic interactions with palmitic (C16:0), oleic (C18:1), linoleic (C18:2) acids and the O/L ratio. Fatty acid levels in these progeny were affected by the parental genotype suggesting that other genes also influence fatty acid composition in peanut. As far as the authors are aware, this is the first study in which all of the nine possible ahFAD2 genotypes were quantitatively measured. CONCLUSIONS: The inheritance of the high oleic trait initially was suggested to be controlled by dominant gene action from two homoeologous genes (ahFAD2A and ahFAD2B) exhibiting complete recessivity. Analyzing the ahFAD2 genotypes and fatty acid compositions of these segregating peanut populations clearly demonstrated that the fatty acid contents are quantitative in nature although much of the variability in the predominant fatty acids (oleic, linoleic, and palmitic) is controlled by only two loci.

Major QTLs for Resistance to Early and Late Leaf Spot Diseases Are Identified on Chromosomes 3 and 5 in Peanut (Arachis hypogaea).

Early and late leaf spots (LLSs) are the major foliar diseases of peanut responsible for severely decreased yield in the absence of intensive fungicide spray programs. Pyramiding host resistance to leaf spots in elite cultivars is a sustainable solution to mitigate the diseases. In order to determine the genetic control of leaf spot disease resistance in peanut, a recombinant inbred line population (Florida-07 × GP-NC WS16) segregating for resistance to both diseases was used to construct a SNP-based linkage map consisting of 855 loci. QTL mapping revealed three resistance QTLs for LLS qLLSA05 (phenotypic variation explained, PVE = 7-10%), qLLSB03 (PVE = 5-7%), and qLLSB05 (PVE = 15-41%) that were consistently expressed over multi-year analysis. Two QTL, qLLSA05 and qLLSB05, confirmed our previously published QTL-seq results. For early leaf spot, three resistance QTLs were identified in multiple years, two on chromosome A03 (PVE = 8-12%) and one on chromosome B03 (PVE = 13-20%), with the locus qELSA03_1.1 coinciding with the previously published genomic region for LLS resistance in GPBD4. Comparative analysis of the genomic regions spanning the QTLs suggests that resistance to early and LLSs are largely genetically independent. In addition, QTL analysis on yield showed that the presence of resistance allele in qLLSB03 and qLLSB05 loci might result in protection from yield loss caused by LLS disease damage. Finally, post hoc analysis of the RIL subpopulation that was not utilized in the QTL mapping revealed that the flanking markers for these QTLs can successfully select for resistant and susceptible lines, confirming the effectiveness of pyramiding these resistance loci to improve host-plant resistance in peanut breeding programs using marker-assisted selection.

Mapping Late Leaf Spot Resistance in Peanut (Arachis hypogaea) Using QTL-seq Reveals Markers for Marker-Assisted Selection.

Late leaf spot (LLS; Cercosporidium personatum) is a major fungal disease of cultivated peanut (Arachis hypogaea). A recombinant inbred line population segregating for quantitative field resistance was used to identify quantitative trait loci (QTL) using QTL-seq. High rates of false positive SNP calls using established methods in this allotetraploid crop obscured significant QTLs. To resolve this problem, robust parental SNPs were first identified using polyploid-specific SNP identification pipelines, leading to discovery of significant QTLs for LLS resistance. These QTLs were confirmed over 4 years of field data. Selection with markers linked to these QTLs resulted in a significant increase in resistance, showing that these markers can be immediately applied in breeding programs. This study demonstrates that QTL-seq can be used to rapidly identify QTLs controlling highly quantitative traits in polyploid crops with complex genomes. Markers identified can then be deployed in breeding programs, increasing the efficiency of selection using molecular tools. Key Message: Field resistance to late leaf spot is a quantitative trait controlled by many QTLs. Using polyploid-specific methods, QTL-seq is faster and more cost effective than QTL mapping.

Genome-wide SNP Genotyping Resolves Signatures of Selection and Tetrasomic Recombination in Peanut.

Peanut (Arachis hypogaea; 2n = 4x = 40) is a nutritious food and a good source of vitamins, minerals, and healthy fats. Expansion of genetic and genomic resources for genetic enhancement of cultivated peanut has gained momentum from the sequenced genomes of the diploid ancestors of cultivated peanut. To facilitate high-throughput genotyping of Arachis species, 20 genotypes were re-sequenced and genome-wide single nucleotide polymorphisms (SNPs) were selected to develop a large-scale SNP genotyping array. For flexibility in genotyping applications, SNPs polymorphic between tetraploid and diploid species were included for use in cultivated and interspecific populations. A set of 384 accessions was used to test the array resulting in 54 564 markers that produced high-quality polymorphic clusters between diploid species, 47 116 polymorphic markers between cultivated and interspecific hybrids, and 15 897 polymorphic markers within A. hypogaea germplasm. An additional 1193 markers were identified that illuminated genomic regions exhibiting tetrasomic recombination. Furthermore, a set of elite cultivars that make up the pedigree of US runner germplasm were genotyped and used to identify genomic regions that have undergone positive selection. These observations provide key insights on the inclusion of new genetic diversity in cultivated peanut and will inform the development of high-resolution mapping populations. Due to its efficiency, scope, and flexibility, the newly developed SNP array will be very useful for further genetic and breeding applications in Arachis.

Compositional and sensory comparisons between normal- and high-oleic peanuts.

The high-oleic-acid trait improves the oxidative stability of peanuts (Arachis hypogaea L.) and their products. The explicit effect of the trait on sensory quality, particularly on off-flavors associated with oil rancidity, has not been well documented. To assess the effect of the trait on off-flavors, data from two independent databases were analyzed to compare sensory quality and composition in normal- versus high-oleic peanut genotypes. In data collected using a sensory panel in the Department of Food Science at North Carolina State University, there were small differences between near-isogenic lines for intensities of the roasted peanut, astringent, over-roast, and nutty attributes, with the high-oleic lines exhibiting slightly greater intensities of those attributes. There were no differences for off-flavors such as fruity, painty, stale, moldy, or petroleum. In data collected from the multistate Uniform Peanut Performance Test and evaluated by a panel in the USDA-ARS Market Quality and Handling Research Unit (MQHRU) at Raleigh, NC, there were differences in chemical composition associated with the high-oleic trait, including differences in oil content, tocopherols, and carbohydrates in addition to the expected differences in fatty acid contents. There were small decreases in the intensities of the sensory attributes cardboard and painty associated with the high-oleic trait in the MQHRU data when all high-oleic lines were compared with all normal-oleic lines. Comparison of the near-isogenic pair NC 7 and N00090ol showed differences in oil and glucose contents, but not in sensory attributes. The high-oleic trait does not appear to have a major impact on sensory quality on average, although there were individual instances in which the trait was associated with shifts in sensory attribute intensities that may be perceptible to consumers.

Allergenicity attributes of different peanut market types.

Four different market classes of peanut (Runner, Virginia Spanish, and Valencia) are commonly consumed in Western countries, but for some consumers peanuts are a main cause of food-induced anaphylaxis. Limited information is available on the comparative allergenicity of these distinct market classes. The aim of this study was to compare allergenicity attributes of different peanut cultivars. The protein content and protein profiles were highly comparable for all tested cultivars. All cultivar samples contained the major allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as assessed by SDS-PAGE and RP-HPLC, although some minor differences in major allergen content were found between samples. All samples were reactive in commercial ELISAs for detection and quantification of peanut protein. IgE-binding potency differed between samples with a maximum factor of 2, indicating a highly comparable allergenicity. Based on our observations, we conclude that peanuts from the main market types consumed in Western countries are highly comparable in their allergenicity attributes, indicating that safety considerations with regard to peanut allergy are not dependent on the peanut cultivar in question.

Oil, sugar, and starch characteristics in peanut breeding lines selected for low and high oil content and their combining ability.

Peanut seeds contain approximately 50% oil on a dry weight basis, making them a high fat food. Reduction of the oil content would make peanuts a more desirable food to fat conscious consumers. Removal of existing oil by processing is not feasible for in-shell peanuts, the dominant product of the North Carolina-Virginia area. To reduce oil content in in-shell peanuts, a genetic solution must be found. However, while reduced oil content is a desirable objective, changes in oil must not be accompanied by significant decreases in any of the desirable aspects of peanut flavor. Because the impact of selection for low or high oil on flavor is not known, it would be useful to know in what form dry matter is being stored in the seed, particularly if it is not being stored as oil. Screening of 584 accessions identified two lines (PI 269723 and PI 315608) with high and two (Robusto 2 and Robusto 3) with low oil contents, each pair differing in sugar content. The four parents were crossed in diallel fashion to investigate patterns of inheritance. General combining abilities (GCA) for oil content closely followed values of the parental lines. One low oil parent (Robusto 2) had a correspondingly elevated GCA for sugar content, but neither low oil parent had the effect of elevating starch in progeny. Reciprocal cross differences were found for starch and sugar contents, suggesting influences of cytoplasmic genes on those traits. These lines serve as resource material for researchers interested in the genetic and physiological aspects of the oil-sugar-starch relationship in peanuts.

Selection of alternative genetic sources of large-seed size in Virginia-type peanut: evaluation of sensory, composition, and agronomic characteristics.

Jenkins Jumbo, the ancestral source of large-seed size in the Virginia market type (Arachis hypogaea L.), has been shown to have a deleterious effect on flavor of peanut. The pervasiveness of Jenkins Jumbo in the ancestry of large-seeded germplasm contributes to the generally less intense roasted peanut flavor of U.S. cultivars of the Virginia market type. As a remedy to this problem, alternative sources of large-seed size were sought. Nine large-seeded selections, with NC 7 and Florunner as checks, were tested in replicated trials in North Carolina and Florida from 1996 to 1998. Pod yield, grade, weight of 100 seeds, and oil, sugar, and starch contents were measured. A descriptive sensory panel evaluated flavor attributes of a roasted sound mature kernel (SMK) sample from each plot. NC 7 scored low for sweet sensory attribute, high for bitter, and median for roasted peanut. UF714021, a multiline incorporating the Altika cultivar with several sister lines, had the best flavor profile of the large-seeded selections, but it did not have particularly large seeds relative to NC 7. The largest seeded selections were X90037 and X90053, both derived from Japan Jumbo. Flavor scores for X90037 were similar to those for NC 7 for roasted peanut (3.0 vs 2.9 flavor intensity units, fiu) and sweet (2.7 vs 2.6 fiu) but worse than NC 7 for bitter (3.3 vs 3.7 fiu) and astringent (3.5 vs 3.7 fiu). X90053 had intermediate values for roasted peanut and astringent, high value for sweet, and low for bitter. Other lines that had or were likely to have Jenkins Jumbo as a recent ancestor were generally poor in roasted flavor, supporting the hypothesis that ancestry from Jenkins Jumbo imparts poor flavor characteristics. With the exception of the unexpected relationship between astringent attribute and extra large kernel (ELK) content (r = 0.82, P < 0.01), there were no significant correlations between sensory attributes and the important agronomic traits: yield, meat, and ELK content. Among the nine large-seeded lines tested in this study, three appear to have greater potential for use as parents: 86x45B-10-1-2-2-b2-B, UF714021, and X90053.

Effect of high-oleic trait and paste storage variables on sensory attribute stability of roasted peanuts.

There has been much interest in the effect of the high-oleic acid trait of peanuts on various quality factors since discovery of high levels of oleic acid in a peanut mutant genotype. The trait provides greater oxidative stability for the high-oleic oil and seed. Several research groups have investigated high-oleic peanut oil and roasted peanut flavor characteristics, which were similar within high-oleic lines compared to Florunner. It was observed that some high-oleic lines derived from the Sunrunner cultivar have consistently higher predicted breeding values for roasted peanut attribute than Sunrunner itself. This study investigated if this apparent effect of the trait was an artifact arising from the handling procedures during processing and storage or from flavor fade. High-oleic lines used were derived by backcrossing the trait into existing cultivars, and the comparison of sensory attribute intensity was with the recurrent parent used in backcrossing. Previous comparisons have been between lines differing in more than just oleate content, that is, with widely different background genotypes that could contribute to the differences observed. Differential rates of change in sensory attributes were found in different background genotypes, suggesting that the comparison of high- and normal-oleic lines should be made in common background genotypes as well as in common production and postharvest environments. There was no measurable change in roasted peanut attribute in samples stored at -20 degrees C over the 63 day duration of this experiment. There were changes in roasted peanut in samples stored at 22 degrees C, confirming that storage at -20 degrees C is sufficient for large studies that require multiple sensory panel sessions over a period of weeks.

Effect of the high-oleic trait on roasted peanut flavor in backcross-derived breeding lines.

The high-oleic trait of peanut (Arachis hypogaea L.) has been suggested to have a positive impact on the roasted peanut sensory attribute. A series of lines derived by backcrossing the high-oleic trait into several existing cultivars were compared with their parent cultivars at locations in Florida, Georgia, North Carolina, and Texas. Breeders grew their high-oleic lines and parents in three-replicate tests at one or two locations. The Florida high-oleic line F435-2-3-B-2-1-b4-B-B-3-b3-b3-1-B was grown at each location. The test included normal- and high-oleic variants of F435, GK 7, NC 7, NC 9, Sunrunner, Tamrun 96, and Tamspan 90. Sound-mature kernel samples were roasted, ground into paste, and evaluated by a sensory panel using a 14-point flavor intensity unit (fiu) scale. Background genotype had an effect (P < 0.01) on the heritable sensory attributes roasted peanut, sweet, and bitter. Oleate level had a positive effect on roasted peanut intensity, increasing it by 0.3 fiu averaged across all seven background genotypes. However, the magnitude of improvement varied across background genotypes. The high-oleic trait had no effect or increased the intensity of the roasted peanut attribute in each background genotype. The increase was greatest in Tamrun 96 (+0.6 fiu, P < 0.05) and Spanish genotypes Tamspan 90 (+0.4 fiu, P < 0.05) and F435 (+0.4 fiu, P < 0.10). A change of 0.5 fiu or more should be perceptible to consumers. Interaction between oleate level and background genotype was detected for sweet (P < 0.10) and bitter (P < 0.01) attributes. The trait had an increasing effect on the bitter attribute only in the background genotype of Tamspan 90 (+0.7 fiu, P < 0.01). There was a nonsignificant increase in bitterness in the other Spanish background genotype, F435. Changes in bitterness in runner- and Virginia-type backgrounds were close to zero. Incorporation of the high-oleic trait into peanut cultivars is likely to improve the intensity of the roasted peanut attribute, but it may also increase the bitter attribute in Spanish genotypes.