Allotetraploid Peanut Research Articles

Genome-wide characterization of phospholipase D family genes in allotetraploid peanut and its diploid progenitors revealed their crucial roles in growth and abiotic stress responses.

Abiotic stresses such as cold, drought and salinity are the key environmental factors that limit the yield and quality of oil crop peanut. Phospholipase Ds (PLDs) are crucial hydrolyzing enzymes involved in lipid mediated signaling and have valuable functions in plant growth, development and stress tolerance. Here, 22, 22 and 46 PLD genes were identified in Arachis duranensis, Arachis ipaensis and Arachis hypogaea, respectively, and divided into α, β, γ, δ, ε, ζ and φ isoforms. Phylogenetic relationships, structural domains and molecular evolution proved the conservation of PLDs between allotetraploid peanut and its diploid progenitors. Almost each A. hypogaea PLD except for AhPLDα6B had a corresponding homolog in A. duranensis and A. ipaensis genomes. The expansion of Arachis PLD gene families were mainly attributed to segmental and tandem duplications under strong purifying selection. Functionally, the most proteins interacting with AhPLDs were crucial components of lipid metabolic pathways, in which ahy-miR3510, ahy-miR3513-3p and ahy-miR3516 might be hub regulators. Furthermore, plenty of cis-regulatory elements involved in plant growth and development, hormones and stress responses were identified. The tissue-specific transcription profiling revealed the broad and unique expression patterns of AhPLDs in various developmental stages. The qRT-PCR analysis indicated that most AhPLDs could be induced by specific or multiple abiotic stresses. Especially, AhPLDα3A, AhPLDα5A, AhPLDβ1A, AhPLDβ2A and AhPLDδ4A were highly up-regulated under all three abiotic stresses, whereas AhPLDα9A was neither expressed in 22 peanut tissues nor induced by any abiotic stresses. This genome-wide study provides a systematic analysis of the Arachis PLD gene families and valuable information for further functional study of candidate AhPLDs in peanut growth and abiotic stress responses.

Gene expression and DNA methylation altering lead to the high oil content in wild allotetraploid peanut (A. monticola).

The wild allotetraploid peanut Arachis monticola contains a higher oil content than the cultivated allotetraploid Arachis hypogaea. Besides the fact that increasing oil content is the most important peanut breeding objective, a proper understanding of its molecular mechanism controlling oil accumulation is still lacking. We investigated this aspect by performing comparative transcriptomics from developing seeds between three wild and five cultivated peanut varieties. The analyses not only showed species-specific grouping transcriptional profiles but also detected two gene clusters with divergent expression patterns between two species enriched in lipid metabolism. Further analysis revealed that expression alteration of lipid metabolic genes with co-expressed transcription factors in wild peanut led to enhanced activity of oil biogenesis and retarded the rate of lipid degradation. In addition, bisulfite sequencing was conducted to characterize the variation of DNA methylation between wild allotetraploid (245, WH 10025) and cultivated allotetraploid (Z16, Zhh 7720) genotypes. CG and CHG context methylation was found to antagonistically correlate with gene expression during seed development. Differentially methylated region analysis and transgenic assay further illustrated that variations of DNA methylation between wild and cultivated peanuts could affect the oil content via altering the expression of peroxisomal acyl transporter protein (Araip.H6S1B). From the results, we deduced that DNA methylation may negatively regulate lipid metabolic genes and transcription factors to subtly affect oil accumulation divergence between wild and cultivated peanuts. Our work provided the first glimpse on the regulatory mechanism of gene expression altering for oil accumulation in wild peanut and gene resources for future breeding applications.

Genome-Wide Identification of Auxin Response Factors in Peanut (Arachis hypogaea L.) and Functional Analysis in Root Morphology

Auxin response factors (ARFs) play important roles in plant growth and development; however, research in peanut (Arachis hypogaea L.) is still lacking. Here, 63, 30, and 30 AhARF genes were identified from an allotetraploid peanut cultivar and two diploid ancestors (A. duranensis and A. ipaensis). Phylogenetic tree and gene structure analysis showed that most AhARFs were highly similar to those in the ancestors. By scanning the whole-genome for ARF-recognized cis-elements, we obtained a potential target gene pool of AhARFs, and the further cluster analysis and comparative analysis showed that numerous members were closely related to root development. Furthermore, we comprehensively analyzed the relationship between the root morphology and the expression levels of AhARFs in 11 peanut varieties. The results showed that the expression levels of AhARF14/26/45 were positively correlated with root length, root surface area, and root tip number, suggesting an important regulatory role of these genes in root architecture and potential application values in peanut breeding.

Chromatin spatial organization of wild type and mutant peanuts reveals high-resolution genomic architecture and interaction alterations

BackgroundThree-dimensional (3D) chromatin organization provides a critical foundation to investigate gene expression regulation and cellular homeostasis.ResultsHere, we present the first 3D genome architecture maps in wild type and mutant allotetraploid peanut lines, which illustrate A/B compartments, topologically associated domains (TADs), and widespread chromatin interactions. Most peanut chromosomal arms (52.3%) have active regions (A compartments) with relatively high gene density and high transcriptional levels. About 2.0% of chromosomal regions switch from inactive to active (B-to-A) in the mutant line, harboring 58 differentially expressed genes enriched in flavonoid biosynthesis and circadian rhythm functions. The mutant peanut line shows a higher number of genome-wide cis-interactions than its wild-type. The present study reveals a new TAD in the mutant line that generates different chromatin loops and harbors a specific upstream AP2EREBP-binding motif which might upregulate the expression of the GA2ox gene and decrease active gibberellin (GA) content, presumably making the mutant plant dwarf.ConclusionsOur findings will shed new light on the relationship between 3D chromatin architecture and transcriptional regulation in plants.

Spontaneous generation of diversity in Arachis neopolyploids (Arachis ipaënsis × Arachis duranensis)4x replays the early stages of peanut evolution.

Polyploidy is considered a driving force in plant evolution and domestication. Although in the genus Arachis, several diploid species were traditionally cultivated for their seeds, only the allotetraploid peanut Arachis hypogaea became the successful, widely spread legume crop. This suggests that polyploidy has given selective advantage for domestication of peanut. Here, we study induced allotetraploid (neopolyploid) lineages obtained from crosses between the peanut’s progenitor species, Arachis ipaënsis and Arachis duranensis, at earlier and later generations. We observed plant morphology, seed dimensions, and genome structure using cytogenetics (FISH and GISH) and SNP genotyping. The neopolyploid lineages show more variable fertility and seed morphology than their progenitors and cultivated peanut. They also showed sexual and somatic genome instability, evidenced by changes of number of detectable 45S rDNA sites, and extensive homoeologous recombination indicated by mosaic patterns of chromosomes and changes in dosage of SNP alleles derived from the diploid species. Genome instability was not randomly distributed across the genome: the more syntenic chromosomes, the higher homoeologous recombination. Instability levels are higher than observed on peanut lines, therefore it is likely that more unstable lines tend to perish. We conclude that early stages of the origin and domestication of the allotetraploid peanut involved two genetic bottlenecks: the first, common to most allotetraploids, is composed of the rare hybridization and polyploidization events, followed by sexual reproductive isolation from its wild diploid relatives. Here, we suggest a second bottleneck: the survival of the only very few lineages that had stronger mechanisms for limiting genomic instability.

Genome-Wide Identification and Characterization of HSP90-RAR1-SGT1-Complex Members From Arachis Genomes and Their Responses to Biotic and Abiotic Stresses

The molecular chaperone complex HSP90-RAR1-SGT1 (HRS) plays important roles in both biotic and abiotic stress responses in plants. A previous study showed that wild peanut Arachis diogoi SGT1 (AdSGT1) could enhance disease resistance in transgenic tobacco and peanut. However, no systematic analysis of the HRS complex in Arachis has been conducted to date. In this study, a comprehensive analysis of the HRS complex were performed in Arachis. Nineteen HSP90, two RAR1 and six SGT1 genes were identified from the allotetraploid peanut Arachis hypogaea, a number close to the sum of those from the two wild diploid peanut species Arachis duranensis and Arachis ipaensis. According to phylogenetic and chromosomal location analyses, thirteen orthologous gene pairs from Arachis were identified, all of which except AhHSP90-A8, AhHSP90-B9, AdHSP90-9, and AiHSP90-9 were localized on the syntenic locus, and they shared similar exon-intron structures, conserved motifs and expression patterns. Phylogenetic analysis showed that HSP90 and RAR1 from dicot and monocot plants diverged into different clusters throughout their evolution. Chromosomal location analysis indicated that AdSGT1 (the orthologous gene of AhSGT1-B3 in this study) might provide resistance to leaf late spot disease dependent on the orthologous genes of AhHSP90-B10 and AhRAR1-B in the wild peanut A. diogoi. Several HRS genes exhibited tissue-specific expression patterns, which may reflect the sites where they perform functions. By exploring published RNA-seq data, we found that several HSP90 genes play major roles in both biotic and abiotic stress responses, especially salt and drought responses. Autoactivation assays showed that AhSGT1-B1 could not be used as bait for yeast two-hybrid (Y2H) library screening. AhRAR1 and AhSGT1 could strongly interact with each other and interact with AhHSP90-B8. The present study represents the first systematic analysis of HRS complex genes in Arachis and provides valuable information for functional analyses of HRS complex genes. This study also offers potential stress-resistant genes for peanut improvement.

Single-cell RNA-seq describes the transcriptome landscape and identifies critical transcription factors in the leaf blade of the allotetraploid peanut (Arachis hypogaea L.).

SummarySingle‐cell RNA‐seq (scRNA‐seq) has been highlighted as a powerful tool for the description of human cell transcriptome, but the technology has not been broadly applied in plant cells. Herein, we describe the successful development of a robust protoplast cell isolation system in the peanut leaf. A total of 6,815 single cells were divided into eight cell clusters based on reported marker genes by applying scRNA‐seq. Further, a pseudo‐time analysis was used to describe the developmental trajectory and interaction network of transcription factors (TFs) of distinct cell types during leaf growth. The trajectory enabled re‐investigation of the primordium‐driven development processes of the mesophyll and epidermis. These results suggest that palisade cells likely differentiate into spongy cells, while the epidermal cells originated earlier than the primordium. Subsequently, the developed method integrated multiple technologies to efficiently validate the scRNA‐seq result in a homogenous cell population. The expression levels of several TFs were strongly correlated with epidermal ontogeny in accordance with obtained scRNA‐seq values. Additionally, peanut AHL23 (AT‐HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 23), which is localized in nucleus, promoted leaf growth when ectopically expressed in Arabidopsis by modulating the phytohormone pathway. Together, our study displays that application of scRNA‐seq can provide new hypotheses regarding cell differentiation in the leaf blade of Arachis hypogaea. We believe that this approach will enable significant advances in the functional study of leaf blade cells in the allotetraploid peanut and other plant species.

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

BackgroundPeanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes.ResultsWe firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation.ConclusionsThis study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

Characterization of mTERF family in allotetraploid peanut and their expression levels in response to dehydration stress

Polyploidization events are prevalent in plants and act as an important evolutionary force for speciation, adaptation and diversification. During this process, the genome undergoes reorganization and large changes. However, the evolutionary characteristics of specific gene families are still not well investigated. Cultivated peanut is an allotetraploid that evolved from a diploid. In this study, we focused on the family of mitochondrial transcription termination factors (mTERF), which is increasing in gene number in higher plants and acts in organellar gene expression. The 65 mTERFs were identified in allotetraploid peanut, but only 31 in diploid peanut, which showed that the genome has not doubled simply during the polyploidization event. Some of the mTERFs may have emerged as a result of reverse complements, deletions, insertions, fusions and so on. mTERFs were divided into three groups according to their predicted localization. Six representative proteins showed their different localization. Most of the AhmTERF mRNAs were detected in root and leaf samples and responded to dehydration stress. Our data provide insights into the evolution of the mTERF gene family in peanut and further support the hypothesis that these genes respond to dehydration stress.

Inheritance of a Novel Heterozygous Peanut Mutant, 5-Small Leaflet

ABSTRACT An unusual 5-Small Leaflet mutant plant was found within the ‘Georgia Green' runner-type peanut (Arachis hypogaea L.) cultivar. Subsequent selfing has not established a true-breeding 5-Small Leaflet genotype. It continues to segregate normal and 5-Small Leaflet plants but with a reduced number of normal leaf plants upon selection for 5-Small Leaflet phenotypes after several self-generations. F1, F2, F3, and F4 data suggests that the 5-Small Leaflet trait is dominant or possibly pseudo-dominant. Likewise, the 5-Small Leaflet mutant can only be used as a pollen parent in crosses, and it has approximately a 1:1 ratio of elongated to normal stigmas, respectively, on individual plants. This is an example of a novel heterozygous peanut mutant plant found within the cultivated allotetraploid peanut.

English

To broaden the genetic base of the allotetraploid peanut (Arachis hypogaea L.), pre-breeding programs have produced interspecific synthetic allotetraploids resulting from the chromosome duplication of hybrids between peanut related diploid species. These allotetraploids were highly cross-fertile with peanut making it possible to access the extensive genetic variability harbored by the wild species. This study aims to evaluate the impact of polyploidization and hybridization in resveratrol content in Arachis hybrids. Resveratrol is a potent antioxidant that has been shown to be useful in the treatment of many human diseases. For that, resveratrol was characterized in five synthetic allotetraploids of wild Arachis, six diploid wild species, three cultivars of A. hypogaea and three backcross (BC) hybrids between synthetic allotetraploids and A. hypogaea. Leaves from these genotypes were ultraviolet (UV) light irradiated for 2 h 30 min and their resveratrol contents were determined by high performance liquid chromatograph (HPLC). Resveratrol was found in all genotypes, but at variable concentrations. Synthetic allotetraploids and peanut did not differ and diploid species had the lowest resveratrol content. The highest concentrations were observed in hybrids between allotetraploids and cultivars of A. hypogaea that were probably the most heterozygous among the genotypes analyzed since their chromosome sets came from different species. This study data suggest a positive effect of polyploidy and hybridization in resveratrol content. Key words: Peanut, wild relatives, polyploidy, pre-breeding.

Comparison of Arachis monticola with Diploid and Cultivated Tetraploid Genomes Reveals Asymmetric Subgenome Evolution and Improvement of Peanut.

Like many important crops, peanut is a polyploid that underwent polyploidization, evolution, and domestication. The wild allotetraploid peanut species Arachis monticola (A. monticola) is an important and unique link from the wild diploid species to cultivated tetraploid species in the Arachis lineage. However, little is known about A. monticola and its role in the evolution and domestication of this important crop. A fully annotated sequence of ≈2.6 Gb A. monticola genome and comparative genomics of the Arachis species is reported. Genomic reconstruction of 17 wild diploids from AA, BB, EE, KK, and CC groups and 30 tetraploids demonstrates a monophyletic origin of A and B subgenomes in allotetraploid peanuts. The wild and cultivated tetraploids undergo asymmetric subgenome evolution, including homoeologous exchanges, homoeolog expression bias, and structural variation (SV), leading to subgenome functional divergence during peanut domestication. Significantly, SV‐associated homoeologs tend to show expression bias and correlation with pod size increase from diploids to wild and cultivated tetraploids. Moreover, genomic analysis of disease resistance genes shows the unique alleles present in the wild peanut can be introduced into breeding programs to improve some resistance traits in the cultivated peanuts. These genomic resources are valuable for studying polyploid genome evolution, domestication, and improvement of peanut production and resistance.

Sequencing of Cultivated Peanut, Arachis hypogaea, Yields Insights into Genome Evolution and Oil Improvement

Cultivated peanut (Arachis hypogaea) is an allotetraploid crop planted in Asia, Africa, and America for edible oil and protein. To explore the origins and consequences of tetraploidy, we sequenced the allotetraploid A. hypogaea genome and compared it with the related diploid Arachis duranensis and Arachis ipaensis genomes. We annotated 39 888 A-subgenome genes and 41 526 B-subgenome genes in allotetraploid peanut. The A. hypogaea subgenomes have evolved asymmetrically, with the B subgenome resembling the ancestral state and the A subgenome undergoing more gene disruption, loss, conversion, and transposable element proliferation, and having reduced gene expression during seed development despite lacking genome-wide expression dominance. Genomic and transcriptomic analyses identified more than 2 500 oil metabolism-related genes and revealed that most of them show altered expression early in seed development while their expression ceases during desiccation, presenting a comprehensive map of peanut lipid biosynthesis. The availability of these genomic resources will facilitate a better understanding of the complex genome architecture, agronomically and economically important genes, and genetic improvement of peanut.

Evidence of Genomic Exchanges between Homeologous Chromosomes in a Cross of Peanut with Newly Synthetized Allotetraploid Hybrids.

Cultivated peanut and synthetics are allotetraploids (2n = 4x = 40) with two homeologous sets of chromosomes. Meiosis in allotetraploid peanut is generally thought to show diploid-like behavior. However, a recent study pointed out the occurrence of recombination between homeologous chromosomes, especially when synthetic allotetraploids are used, challenging the view of disomic inheritance in peanut. In this study, we investigated the meiotic behavior of allotetraploid peanut using 380 SSR markers and 90 F2 progeny derived from the cross between Arachis hypogaea cv Fleur 11 (AABB) and ISATGR278-18 (AAKK), a synthetic allotetraploid that harbors a K-genome that was reported to pair with the cultivated B-genome during meiosis. Segregation analysis of SSR markers showed 42 codominant SSRs with unexpected null bands among some progeny. Chi-square tests for these loci deviate from the expected 1:2:1 Mendelian ratio under disomic inheritance. A linkage map of 357 codominant loci aligned on 20 linkage groups (LGs) with a total length of 1728 cM, averaging 5.1 cM between markers, was developed. Among the 10 homeologous sets of LGs, one set consisted of markers that all segregated in a polysomic-like pattern, six in a likely disomic pattern and the three remaining in a mixed pattern with disomic and polysomic loci clustered on the same LG. Moreover, we reported a substitution of homeologous chromosomes in some progeny. Our results suggest that the homeologous recombination events occurred between the A and K genomes in the newly synthesized allotetraploid and have been highlighted in the progeny. Homeologous exchanges are rarely observed in tetraploid peanut and have not yet been reported for AAKK and AABB genomes. The implications of these results on peanut breeding are discussed.

Genomics: Decoding the ancestors of peanut.

Cultivated peanut has a large, complex genome, so obtaining its entire sequence is challenging. De novo assemblies of two diploid ancestor genomes provide high-quality reference sequences for decoding allotetraploid peanut genomes, and will become valuable resources for breeding and evolutionary studies.

Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut.

Molecular cloning and characterization of an acyl-ACP thioesterase gene (AhFatB1) from allotetraploid peanut (Arachis hypogaea L.)

Acyl-acyl carrier protein (ACP) thioesterase is a nuclear encoded plastid localized enzyme which plays an essential role in chain termination during de novo fatty acid synthesis in plant. FatB genes coding for this enzyme from a variety of plant species have been isolated and characterized. However, there are few reports on such genes in peanut ( Arachis hypogaea ), an important edible and oilseed crop. In this study, full-length cDNA of an acyl-acyl carrier protein thioesterase (EC 3.1.2.14), designated as AhFatB1 , was isolated from peanut cDNA libraries. The putative open reading frames consist of 1239 bp with five introns spliced from the corresponding genomic sequence, encoding a 413 amino acid protein, two homologous genes, AhFatB1A and AhFatB1B , with sequence difference at the 5’ non-coding regions were characterized at the nucleotide level from different cultivated peanut genotypes, and the two genes have their origin in different diploid progenitor which was evidenced by the characterization of AhFatB1 genes from Arachis duranensis and Arachis ipaensis , the putative A-genome donor and B-genome donor respectively. AhFatB1 genes are constitutively expressed in peanut tissues and the total FatB1 transcript accumulations are temporally regulated during peanut seed development. Keywords: Peanut thioesterase, palmitic acid oilseed

A high-density genetic map of Arachis duranensis, a diploid ancestor of cultivated peanut

BackgroundCultivated peanut (Arachis hypogaea) is an allotetraploid species whose ancestral genomes are most likely derived from the A-genome species, A. duranensis, and the B-genome species, A. ipaensis. The very recent (several millennia) evolutionary origin of A. hypogaea has imposed a bottleneck for allelic and phenotypic diversity within the cultigen. However, wild diploid relatives are a rich source of alleles that could be used for crop improvement and their simpler genomes can be more easily analyzed while providing insight into the structure of the allotetraploid peanut genome. The objective of this research was to establish a high-density genetic map of the diploid species A. duranensis based on de novo generated EST databases. Arachis duranensis was chosen for mapping because it is the A-genome progenitor of cultivated peanut and also in order to circumvent the confounding effects of gene duplication associated with allopolyploidy in A. hypogaea.ResultsMore than one million expressed sequence tag (EST) sequences generated from normalized cDNA libraries of A. duranensis were assembled into 81,116 unique transcripts. Mining this dataset, 1236 EST-SNP markers were developed between two A. duranensis accessions, PI 475887 and Grif 15036. An additional 300 SNP markers also were developed from genomic sequences representing conserved legume orthologs. Of the 1536 SNP markers, 1054 were placed on a genetic map. In addition, 598 EST-SSR markers identified in A. hypogaea assemblies were included in the map along with 37 disease resistance gene candidate (RGC) and 35 other previously published markers. In total, 1724 markers spanning 1081.3 cM over 10 linkage groups were mapped. Gene sequences that provided mapped markers were annotated using similarity searches in three different databases, and gene ontology descriptions were determined using the Medicago Gene Atlas and TAIR databases. Synteny analysis between A. duranensis, Medicago and Glycine revealed significant stretches of conserved gene clusters spread across the peanut genome. A higher level of colinearity was detected between A. duranensis and Glycine than with Medicago.ConclusionsThe first high-density, gene-based linkage map for A. duranensis was generated that can serve as a reference map for both wild and cultivated Arachis species. The markers developed here are valuable resources for the peanut, and more broadly, to the legume research community. The A-genome map will have utility for fine mapping in other peanut species and has already had application for mapping a nematode resistance gene that was introgressed into A. hypogaea from A. cardenasii.

TILLING for allergen reduction and improvement of quality traits in peanut (Arachis hypogaea L.)

BackgroundAllergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut.ResultsTwo similar copies of a major allergen gene, Ara h 1, have been identified in tetraploid peanut, one in each subgenome. The same situation has been shown for major allergen Ara h 2. Due to the challenge of discriminating between homeologous genes in allotetraploid peanut, nested PCR was employed, in which both gene copies were amplified using unlabeled primers. This was followed by a second PCR using gene-specific labeled primers, heteroduplex formation, CEL1 nuclease digestion, and electrophoretic detection of labeled fragments. Using ethyl methanesulfonate (EMS) as a mutagen, a mutation frequency of 1 SNP/967 kb (3,420 M2 individuals screened) was observed. The most significant mutations identified were a disrupted start codon in Ara h 2.02 and a premature stop codon in Ara h 1.02. Homozygous individuals were recovered in succeeding generations for each of these mutations, and elimination of Ara h 2.02 protein was confirmed. Several Ara h 1 protein isoforms were eliminated or reduced according to 2D gel analyses. TILLING also was used to identify mutations in fatty acid desaturase AhFAD2 (also present in two copies), a gene which controls the ratio of oleic to linoleic acid in the seed. A frameshift mutation was identified, resulting in truncation and inactivation of AhFAD2B protein. A mutation in AhFAD2A was predicted to restore function to the normally inactive enzyme.ConclusionsThis work represents the first steps toward the goal of creating a peanut cultivar with reduced allergenicity. TILLING in peanut can be extended to virtually any gene, and could be used to modify other traits such as nutritional properties of the seed, as shown in this study.

Identification and Molecular Characterization of Homeologous Δ9-Stearoyl Acyl Carrier Protein Desaturase 3 Genes from the Allotetraploid Peanut (Arachis hypogaea)

The stearoyl–acyl carrier protein (ACP) desaturase (SAD) is a nuclear-encoded, plastid-localized soluble desaturase that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays a key role in the determination of the properties of the majority of cellular glycerolipids. Sad genes from a variety of plant species have been cloned and characterized. However, in peanut (Arachis hypogaea), an important edible and oilseed crop, these genes have not yet been characterized. By searching peanut expressed sequence tag (EST) and parallel sequencing (454) libraries, we have identified three members of the ahSad gene family. Among them, only one gene, ahSad3, was exclusively expressed during seed development and in a manner fully corresponding to oil accumulation. Both ahSad3 homeologous genes (ahSad3A and ahSad3B) were recovered from the allotetraploid peanut, and their mRNA expression levels were characterized. The open reading frames for ahSad3A and ahSad3B are 98% identical and consist of 1,158 bp, encoding a 386-full-amino-acid protein, with one intron in the coding sequence. Comparisons of the sequences of these two homeologous genes revealed seven single-nucleotide polymorphisms and one triplet insertion in the coding region. Southern blot analysis indicated that there are only two copies of the ahSad3 gene in the peanut genome. Homeolog-specific gene expression analysis showed that both ahSad3 homeologs are expressed in developing seeds, but gene expression is significantly biased toward the B genome. Our results point to ahSad3 as a possible target gene for manipulation of fatty acid saturation in A. hypogaea.