Kelly D. Chamberlin

Development of a real-time PCR genotyping assay to identify high oleic acid peanuts ( Arachis hypogaea L.)

Oleic acid, a monounsaturated, omega-9 fatty acid found in peanut (Arachis hypogaea L.) oil is an important seed quality trait because it provides increased shelf life, improved flavor, enhanced fatty acid composition, and has a beneficial effect on human health. Hence, a concentrated effort has been put forth on developing peanut cultivars that have high oleic acid (>74%) and a low amount (<10%) of linoleic acid, a polyunsaturated omega-6 fatty acid. A main bottleneck, however, in breeding research is fast selection of the trait(s) of interest. Therefore, in an effort to expedite breeding efforts, a real-time PCR genotyping assay was developed to rapidly identify the wild type and the mutant allele that are responsible for normal or high levels of oleic acid, respectively in peanut seeds. This test utilizes two TaqMan® probes to detect the presence of an indel (insertion/deletion) in FAD2B and can be employed on DNA extracted from either seeds or leaves. The presence of the insertion (mutant allele) in fad2B causes a frameshift downstream in the coding sequence that ultimately alters the mRNA transcript level, and thus, decreases the activity of microsomal oleoyl-PC desaturase enzyme which converts oleic acid (C18:1) to linoleic acid (C18:2). Validation of the real-time assay was carried out by quantitatively evaluating the fatty acid composition by gas chromatography (GC). Overall, this real-time PCR assay facilitates the identification of progeny carrying the high oleic acid alleles, and thus, allows early elimination of undesirable non-high oleic acid lines in segregating populations.

Evaluation of the U.S. peanut mini core collection using a molecular marker for resistance to Sclerotinia minor Jagger

Cultivated peanut, the second most economically important legume crop throughout the United States and the third most important oilseed in the world, is consistently threatened by various diseases and pests. Sclerotinia minor Jagger (S. minor), the causal agent of Sclerotinia blight, is a major threat to peanut production in the Southwestern U.S., Virginia, and North Carolina and can reduce yield by up to 50% in severely infested fields. Although host plant resistance would provide the most effective solution to managing Sclerotinia blight, limited sources of resistance to the disease are available for use in breeding programs. Peanut germplasm collections are available for exploration and identification of new sources of resistance, but traditionally the process is lengthy, requiring years of field testing before those potential sources can be identified. Molecular markers associated with phenotypic traits can speed up the screening of germplasm accessions, but until recently none were available for Sclerotinia blight resistance in peanut. This study objective of this study was to characterize the US peanut mini-core collection with regards to a recently discovered molecular marker associated with Sclerotinia blight resistance. Ninety-six accessions from the collection were available and genotyped using the SSR marker and 39 total accessions from spanish, valencia, runner market types were identified as new potential sources of resistance and targeted for further evaluation in field tests for Sclerotinia blight resistance.

Reaction of the Core Collection of Peanut Germplasm to Sclerotinia Blight and Pepper Spot 1

In 2001, entries from the peanut core collection, a subset of the USDA peanut germplasm collection, were planted in non-replicated plots in a field with a history of Sclerotinia blight caused by Sclerotinia minor. Variability existed among entries for reaction to Sclerotinia blight. Of the 744 entries evaluated, 11% had no disease, nearly 30% had ,10% disease incidence, and only 21% had 50% disease incidence or more. Most of the resistant entries had an upright growth habit and were in early and mid-maturity groups. Many of the early maturing entries were susceptible to the foliar disease pepper spot which occurred throughout the study. Entries were selected for further evaluation in replicated plots based on a nil to low (,10%) incidence of Sclerotinia blight, adaptation and/or vigor, and other desirable characteristics such as an intermediate to prostrate growth habit and pepper spot resistance. Selected entries were retested in both 2002 and 2003 (n 5 62) and compared to resistant (Tamspan 90), moderately resistant (Tamrun 96), and susceptible (Okrun) reference cultivars. Most entries (55 in 2001 and 46 in 2003) had disease incidence less than Tamrun 96 and similar to Tamspan 90. In 2003 when disease incidence was highest, all 46 entries with resistant reactions similar to that of Tamspan 90 had erect plant growth habits except for entries 208 and 582 which were prostrate, and entries 273, 128, and 804 which were intermediate. Resistance to Sclerotinia blight and yield similar to Tamspan 90, plant habit, and/or reactions to pepper spot and web blotch were used to select the best entries. Entries 208, 128, 804, 582, and 273 combined resistance to Sclerotinia blight, pepper spot, and web blotch with less than erect growth habits. Entry 103 had good Sclerotinia blight resistance and yield, but an upright growth habit. Entry 92 had an upright growth habit and low yield, but good Sclerotinia blight resistance. Entries 92 and 103 had upright growth habits but were among the best entries for resistant to pepper spot and web blotch. Entries 426, 184, and 562 were upright and susceptible to pepper spot, but had resistance to web blotch and the best resistance to Sclerotinia blight. These entries appear to be useful sources of resistance to Sclerotinia blight for breeding programs and for increasing the probability of finding additional sources of resistance in clusters of germplasm identified within the entire USDA collection.

Genotyping and Fatty Acid Composition Analysis in Segregating Peanut (Arachis hypogaea L.) Populations

Abstract Oleic acid (C18∶1), a monounsaturated omega-9 fatty acid, is an important seed quality trait in peanut (Arachis hypogaea L.) because it provides enhanced fatty acid composition, a beneficial effect on human health, improved flavor, and increased shelf life for stored food products by delaying rancidity. Consequently, an emphasis has been placed on breeding peanuts with high levels of oleic acid and low levels of linoleic acid (C18∶2), a polyunsaturated, omega-6 fatty acid. Therefore, crosses were prepared between high oleic and normal peanut lines to develop segregating F2 populations. Total fatty acid composition and the ahFAD2B genotype were determined in the parents and progeny. The oleic to linoleic (O/L) ratio varied from 0.85 to 30.30 in the F2 progeny. Comparing the mean oleic acid values from the three genotypic classes (Ol2Ol2, Ol2ol2, and ol2ol2) in each population confirmed that the means were significantly different. Statistical analysis demonstrated that oleic acid was negatively cor...

Three Inoculation Methods for Evaluating Sclerotinia Blight Resistance in Peanut

ABSTRACT Laboratory-based assays for screening germplasm for resistance to Sclerotinia blight in peanuts can be conducted year-round, and thus may accelerate progress in breeding for resistant plants. Three previously proposed inoculation methods (using main stems of intact plants, detached main stems, or detached leaflets) were compared on six peanut genotypes known to represent range of resistance to Sclerotinia blight in the field or laboratory. The intact plant and detached main stem assays identified the most resistant and susceptible genotypes, but different results were obtained from either assay with Red River Runner, a cultivar with intermediate resistance to Sclerotinia blight. No differences among genotypes were observed with the detached leaflet assay. The sensitivity ratio was used to compare the three inoculation methods and identified the intact plant assay as the method with the smallest error variance. These results help identify the most efficient method for assaying physiological resist...

Genotyping of the Valencia Peanut Core Collection with a Molecular Marker Associated with Sclerotinia blight Resistance

ABSTRACT Cultivated peanut, the second most economically important legume crop throughout the United States and the third most important oilseed in the world, is consistently threatened by various diseases and pests. Sclerotinia blight, (causal agents Sclerotinia sclerotiorum (S. sclerotiorum) and Sclerotinia minor Jagger (S. minor))is a major threat to peanut production in the Southwestern U.S., Virginia, and North Carolina and can reduce yield by up to 50% in severely infested fields. S. sclerotiorum has now been reported in areas of eastern New Mexico and west Texas where all U.S. grown Valencia peanuts are produced, commonly in organic cropping environments. Host plant resistance provides the most effective solution to managing Sclerotinia blight, especially in organic systems where pesticide use is not an option for disease control. To date, no Valencia cultivars with Sclerotinia blight resistance have been released. In this study, the Valencia peanut core germplasm collection was genotyped with a Si...

Lignin Content and Resistance to Sclerotinia minor in Peanut

ABSTRACT Lignin has been shown to be an important component for plant defense in several pathosystems, but the relationship between peanut stem lignin content and resistance in the field to Sclerotinia blight has not been investigated. Stem lignin was quantified from twenty runner, six virginia, and ten spanish genotypes grown in the greenhouse using the acetyl bromide method. Significant differences in lignin content were found within the runner and spanish entries, but not among the virginia genotypes. Disease data collected in the field over two to three years were used to test correlations between lignin content and Sclerotinia blight resistance for a subset of the runner and virginia entries. No significant correlations were found. Within the runner entries, the highest and lowest stem lignin content was found in entries with the most disease resistance. These results indicate that preformed stem lignin content is not a reliable predictor for resistance to Sclerotinia blight in peanut. In addition, c...

Response to oxalic acid as a resistance assay for Sclerotinia minor in peanut

ABSTRACT Response to oxalic acid was evaluated as an assay for screening peanut breeding lines for resistance to Sclerotinia blight caused by Sclerotinia minor. Detached stems of six runner- and seven spanish-type peanut genotypes, varying in resistance to Sclerotinia blight, were placed in 0, 20, and 50 mM oxalic acid. Wilt severity and length of stem discoloration were rated 12, 18, and 24 hr after immersion. Wilt and stem discoloration were not observed on stems placed in water (0 mM), but differences among entries were observed for some time intervals when placed in the 20 and 50 mM oxalic acid solutions. Susceptible runner cultivars exhibited the most severe wilt symptoms, but few differences in wilting were found among the spanish genotypes. More differences in stem discoloration were observed among the spanish peanuts than the runner entries, but length of discoloration did not appear to be correlated with field resistance. Oxalic acid appears to have some potential as a resistance assay for Sclero...

Deployment: Regulations and Steps for Commercialization

Genetically modified (GM) crops produced by genetic engineering continue to increase in production worldwide. In 2007, 23 countries produced 282.3 million acres of GM crops, up 30.3 million acres since 2006 [International Service of AgriBiotech Applications (ISAAA)]. The US ranks first in GM crop production, followed by Argentina, Brazil and Canada, in that order. The potential for economic and social gain from the production of GM crops is generally greater in developing countries/economies, since there is usually a higher incidence of disease/pests and larger potential for yield increase (Abdalla et al. 2003; Qaim 2005). According to Nossal et al. (2008), GM crop production in 2007 increased by 20% in countries with emerging economies versus 6% in developed countries.