Peanut Genome Research Articles

Development of three real-time PCR assays to detect peanut allergen residue in processed food products

Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry, that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5 pg peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut traces in a model food product where they could detect 10 mg kg−1 peanut.

Transferability of Soybean SSR Markers in Peanut (Arachis hypogaea L.)

Simple sequence repeats (SSRs) are useful DNA markers in plant genetic research. However, they are not fully exploited in peanut because of the high cost and labor intensity involved in their development. Many studies have showed that DNA markers could be transferable among related species due to the conserved regions in their genomes. The objective of this study was to investigate the transferability of soybean SSR markers to peanut because of the availability of a large number of soybean SSRs. Four hundred thirty-two soybean SSR primer pairs were used to amplify peanut genomic DNAs extracted from four cultivated peanut lines. The result showed that 25% of soybean SSR primer pairs tested in this study could amplify peanut genomic DNA. Among these transferable SSR markers, 28% have detected polymorphism among these peanut lines. These transferable markers will benefit peanut genome research by not only providing additional DNA markers in peanut, but also allowing comparative mapping to be possible between peanut and soybean.

Organization and evolution of resistance gene analogs in peanut

The scarcity of genetic polymorphism in Arachis hypogaea (peanut), as in other monophyletic polyploid species, makes it especially vulnerable to nematode, bacterial, fungal, and viral pathogens. Although no disease resistance genes have been cloned from peanut itself, the conserved motifs in cloned resistance genes from other plant species provide a means to isolate and analyze similar genes from peanut. To survey the number, diversity, evolutionary history, and genomic organization of resistance gene-like sequences in peanut, we isolated 234 resistance gene analogs (RGAs) by using primers designed from conserved regions of different classes of resistance genes including NBS-LRR, and LRR-TM classes. Phylogenetic and sequence analyses were performed to explore evolutionary relationships both among peanut RGAs and with orthologous genes from other plant taxa. Fifty-six overgos designed from the RGA sequences on the basis of their phyletic association were applied to a peanut BAC library; 736 hybridizing BAC clones were fingerprinted and contigs were formed in order to gain insights into the genomic organization of these genes. All the fingerprinting gels were blotted and screened with the respective overgos in order to verify the authenticity of the hits from initial screens, and to explore the physical organization of these genes in terms of both copy number and distribution in the genome. As a result, we identified 250 putative resistance gene loci. A correlation was found between the phyletic positions of the sequences and their physical locations. The BACs isolated here will serve as a valuable resource for future applications, such as map-based cloning, and will help improve our understanding of the evolution and organization of these genes in the peanut genome.

Construction and characterization of a peanut HindIII BAC library

Bacterial artificial chromosome (BAC) libraries have been an essential tool for physical analyses of genomes of many crops. We constructed and characterized the first large-insert DNA library for Arachis hypogaea L. The HindIII BAC library contains 182,784 clones; only 5,484 (3%) had no inserts; and the average insert size is 104.05 kb. Chloroplast DNA contamination was very low, only nine clones, and r-DNA content was 1,208, 0.66% of clones. The depth of coverage is estimated to be 6.5 genome-equivalents, allowing the isolation of virtually any single-copy locus. This rate of coverage was confirmed with the application of 20 overgos, which identified 305 positive clones from the library. The identification of multiple loci by most probes in polyploids complicates anchoring of physical and genetic maps. We explored the practicality of a hybridization-based approach for determination of map locations of BAC clones in peanut by analyzing 94 clones detected by seven different overgos. The banding patterns on Southern blots were good predictors of contig composition; that is, the clones that shared the same size bands and ascribed to the same overgos usually also located in the same contigs. This BAC library has great potential to advance future research about the peanut genome.

Exploratory integration of peanut genetic and physical maps and possible contributions from Arabidopsis

Arachis hypogaea is a widely cultivated crop both as an oilseed and protein source. The genomic analysis of Arachis species hitherto has been limited to the construction of genetic maps; the most comprehensive one contains 370 loci over 2,210 cM in length. However, no attempt has been made to analyze the physical structure of the peanut genome. To investigate the practicality of physical mapping in peanut, we applied a total of 117 oligonucleotide-based probes ("overgos") derived from genetically mapped RFLP probes onto peanut BAC filters containing 182,784 peanut large-insert DNA clones in a multiplex experimental design; 91.5% of the overgos identified at least one BAC clone. In order to gain insights into the potential value of Arabidopsis genome sequence for studies in divergent species with complex genomes such as peanut, we employed 576 Arabidopsis-derived overgos selected on the basis of maximum homology to orthologous sequences in other plant taxa to screen the peanut BAC library. A total of 353 (61.3%) overgos detected at least one peanut BAC clone. This experiment represents the first steps toward the creation of a physical map in peanut and illustrates the potential value of leveraging information from distantly related species such as Arabidopsis for both practical applications such as comparative map-based cloning and shedding light on evolutionary relationships. We also evaluated the possible correlation between functional categories of Arabidopsis overgos and their success rates in hybridization to the peanut BAC library.

A genetic engineering strategy to eliminate peanut allergy

Peanut allergy is an IgE-mediated hypersensitivity reaction with an increasing prevalence worldwide. Despite its seriousness, to date, there is no cure. Genetic engineering strategies can provide a solution. The post-transcriptional gene silencing (PTGS) model can be used effectively to knock out the production of allergenic proteins in peanut by specific degradation of the endogenous target messenger RNA (mRNA). Ara h 2, the most potent peanut allergenic protein, was selected as a model to demonstrate the feasibility of this concept. Transgenic peanut plants were produced via microprojectile-mediated transformation of peanut embryos using a plasmid construct, which contains a fragment of the coding region of Ara h 2 linked to an enhanced CaMV 35S constitutive promoter. Molecular analyses, including polymerase chain reaction and Southern blots, confirmed the presence of the stable integration of the Ara h 2 transgene into the peanut genome. Northern hybridization showed the expression of the Ara h 2 transgene in all vegetative tissues of the mature transgenic peanut plants, indicating the stable expression of the truncated Ara h 2 transgene throughout the development of the plants. It is, therefore, reasonable to expect that the truncated Ara h 2 transgene transcripts will be synthesized in the seeds and will trigger the specific degradation of endogenous Ara h 2 mRNA. The next step will be to grow the transgenic peanut plants to full maturity for seed production and to determine the level of allergen Ara h 2.

Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression

The nucleocapsid protein (N) gene of the lettuce isolate of tomato spotted wilt virus (TSWV) was inserted into peanut (Arachis hypogaea L.) via microprojectile bombardment. Constructs containing the hph gene for resistance to the antibiotic hygromycin and the TSWV N gene were used for bombardment of peanut somatic embryos. High frequencies of transformation and regeneration of plants containing the N gene were obtained. Southern blot analysis of independent transgenic lines revealed that one to several copies of the N gene were integrated into the peanut genome. Northern blot, RT-PCR and ELISA analyses indicated that a gene silencing mechanism may be operating in primary transgenic lines containing multiple copy insertions of the N transgene. One transgenic plant which contained a single copy of the transgene expressed the N protein in the primary transformant, and the progeny segregated in a 3 :1 ratio based upon ELISA determination.

2104 Expression of serotonin N-acetyltransferase mRNA in the rat retina

Genetics and genomics will have the potential to enhance sustainable peanut production. The major contribution of these technologies for peanut will likely be improved disease resistance, oil quality, and enhanced productivity. Those attributes may be achieved more effectively through genomic biotechnology to utilize the genetic resources preserved in germplasm collections for maximizing the genetic potential in breeding. Superior cultivars will maximize desirable genetic traits and provide growers with cultivars that are locally adapted and highly productive. Genomics involves the study of the complete genetic makeup, through mapping, sequencing, and functional studies to identify genes that regulate, control or modify trait expression. With the development in sequencing technologies, next-generation sequencing (NGS) has opened the gateway to sequence the genomes in a fast, reliable, and cost-effective manner. The international peanut genome sequencing project was discussed in 2010 and launched in 2012 after much deliberation and strategic planning. The exciting news is that the Peanut Genome Consortium, representing a coalition of international scientists and stakeholders engaged in the International Peanut Genome Initiative, announced the public release of the first chromosomal-scale draft sequences of two peanut species (Arachis duranensis and Arachis ipaënsis) on April 2, 2014 (http://www.peanutbioscience.com/images/4-2-14_News_Release_Notice.pdf). In this chapter, we update the information on the molecular markers, genetic linkage maps, and quantitative trait loci (QTLs) of traits mapped to date. Information of the preview of genome sequences, which would be available in peanut populations for high resolution genetic and trait mapping such as multiparent advanced generation intercross and nested association mapping, is also discussed. Furthermore, an innovative method to identify a causal mutant single nucleotide polymorphism or major allele for QTLs through NGS techniques such as MutMap and QTLseq is also discussed in the section on emerging trait mapping strategies. A special emphasis on Africa and China is given in different sections to update on the activities in peanut research and emerging issues. The goal of this chapter is to update the new progress in the development of genetic and genomic resources and technologies in peanuts.

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene

2103 Light-induced FOS and junb proteins in the suprachiasmatic nucleus of CS mice during rhythm splitting

Genetics and genomics will have the potential to enhance sustainable peanut production. The major contribution of these technologies for peanut will likely be improved disease resistance, oil quality, and enhanced productivity. Those attributes may be achieved more effectively through genomic biotechnology to utilize the genetic resources preserved in germplasm collections for maximizing the genetic potential in breeding. Superior cultivars will maximize desirable genetic traits and provide growers with cultivars that are locally adapted and highly productive. Genomics involves the study of the complete genetic makeup, through mapping, sequencing, and functional studies to identify genes that regulate, control or modify trait expression. With the development in sequencing technologies, next-generation sequencing (NGS) has opened the gateway to sequence the genomes in a fast, reliable, and cost-effective manner. The international peanut genome sequencing project was discussed in 2010 and launched in 2012 after much deliberation and strategic planning. The exciting news is that the Peanut Genome Consortium, representing a coalition of international scientists and stakeholders engaged in the International Peanut Genome Initiative, announced the public release of the first chromosomal-scale draft sequences of two peanut species (Arachis duranensis and Arachis ipaënsis) on April 2, 2014 (http://www.peanutbioscience.com/images/4-2-14_News_Release_Notice.pdf). In this chapter, we update the information on the molecular markers, genetic linkage maps, and quantitative trait loci (QTLs) of traits mapped to date. Information of the preview of genome sequences, which would be available in peanut populations for high resolution genetic and trait mapping such as multiparent advanced generation intercross and nested association mapping, is also discussed. Furthermore, an innovative method to identify a causal mutant single nucleotide polymorphism or major allele for QTLs through NGS techniques such as MutMap and QTLseq is also discussed in the section on emerging trait mapping strategies. A special emphasis on Africa and China is given in different sections to update on the activities in peanut research and emerging issues. The goal of this chapter is to update the new progress in the development of genetic and genomic resources and technologies in peanuts.

Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens

Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediated transformation system. Leaf section explants were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBI121 containing the genes for β-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Approximately 10% of the shoots regenerated on selection medium were GUS-positive. Five independent transformation events resulted in the production of 52 fertile transgenic peanut plants. On average, 240 d were required between seed germination for explant preparation and the production of mature t1 seed by T0 plants. Molecular analysis of transgenic plants confirmed the stable integration of the transgenes into the peanut genome. GUS expression segregated in a 3∶1 Mendelian ratio in most T1 generation plants.

DNA Content and Heterochromatin Variations in Various Tissues of Peanut (Arachis hypogaea)

The average 2C DNA amount for the peanut (Arachis hypogaea L.) genome is 4.21 pg, and 73% of the dormant peanut cotyledon nuclei displayed 8C DNA amounts or higher, as compared to 0 to 4% in root-shoot apices and leaf tissue. Thermal melt profiles and heterochromatin values indicated replication of the whole genome. Cotyledon nuclear DNA declined in the percent of polyploid nuclei as well as DNA amounts within ploidy classes during germination. The presence of high DNA C levels in cotyledons generated during embryogeny is interpreted to increase the protein-synthesizing capacity and subsequently supplies a ready source of nucleosides and phosphates during early embryo growth as a result of DNA degradation. However, the later DNA decline at the onset of cotyledon senescence was age related similarly to leaf senescence. The change in proportion of heterochromatin was related to the metabolic state of the tissue and not to the DNA content, as dormant and senescing nuclei contained a higher proportion of heterochromatin as compared to nuclei from metabolically active tissue such as germinating roots. The shift in heterochromatin is interpreted to be involved in gene expression.

DNA CONTENT AND HETEROCHROMATIN VARIATIONS IN VARIOUS TISSUES OF PEANUT (ARACHIS HYPOGAEA)

The average 2C DNA amount for the peanut (Arachis hypogaea L.) genome is 4.21 pg, and 73% of the dormant peanut cotyledon nuclei displayed 8C DNA amounts or higher, as compared to 0 to 4% in root‐shoot apices and leaf tissue. Thermal melt profiles and heterochromatin values indicated replication of the whole genome. Cotyledon nuclear DNA declined in the percent of polyploid nuclei as well as DNA amounts within ploidy classes during germination. The presence of high DNA C levels in cotyledons generated during embryogeny is interpreted to increase the protein‐synthesizing capacity and subsequently supplies a ready source of nucleosides and phosphates during early embryo growth as a result of DNA degradation. However, the later DNA decline at the onset of cotyledon senescence was age related similarly to leaf senescence. The change in proportion of heterochromatin was related to the metabolic state of the tissue and not to the DNA content, as dormant and senescing nuclei contained a higher proportion of heterochromatin as compared to nuclei from metabolically active tissue such as germinating roots. The shift in heterochromatin is interpreted to be involved in gene expression.