Ye Chu

The genome sequences of Arachis duranensis and Arachis ipaensis , the diploid ancestors of cultivated peanut

Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ∼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanuts A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanuts subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.

Validation of gel-free, label-free quantitative proteomics approaches: Applications for seed allergen profiling

Plant seeds provide a significant portion of the protein present in the human diet, but are also the major contributors of allergenic proteins that cause a majority of the reported cases of food-induced anaphylaxis. New varieties of grains and nuts as well as other seeds could be screened for allergen content before they are introduced as cultivars for food production using mass spectrometry-based quantitation approaches. Here, we present a practical comparison of gel-free and label-free methods, peak integration and spectral counting, using a linear trap mass spectrometer. The results show that both methods are linear and reproducible with protein standards from 5-200 ng, however, bioinformatic analysis for spectral counting is much simpler and therefore more amenable to high-throughput sample processing. We therefore applied spectral counting towards the analysis of transgenic peanut lines targeting the reduction of a prominent allergen. Spectral count analysis of an Ara h 2 (conglutin-7) RNA-silenced line confirmed reduction of this allergen as well as Ara h 6 (conglutin), which was further confirmed by quantitative immunoblotting. Other collateral changes include an increase in Ara h 10 (oleosin 1) in one of the three lines, a decrease in conarachin as well as increased 13-lipoxygenase and Ahy-3 (arachin) in two of three lines.

Reduction of IgE Binding and Nonpromotion of Aspergillus flavus Fungal Growth by Simultaneously Silencing Ara h 2 and Ara h 6 in Peanut

The most potent peanut allergens, Ara h 2 and Ara h 6, were silenced in transgenic plants by RNA interference. Three independent transgenic lines were recovered after microprojectile bombardment, of which two contained single, integrated copies of the transgene. The third line contained multiple copies of the transgene. Ara h 2 expression was significantly suppressed in all three lines, whereas Ara h 6 was reduced in two lines. Expression of peanut allergens Ara h 1 and Ara h 3 was not noticeably affected. Significant reduction of human IgE binding to Ara h 2 and Ara h 6 also was observed. Seed weight and germination data from transgenic and nontransgenic segregants showed no significant differences. Data collected from in vitro Aspergillus flavus infection indicate no significant difference in fungal growth between the transgenic lines and the nontransgenic controls. These data suggest that silencing Ara h 2 and Ara h 6 is a feasible approach to produce hypoallergenic peanut.

Chromosomal and phylogenetic context for conglutin genes in Arachis based on genomic sequence

AbstractsComparative genomic and cDNA sequence analysis of ara h 2, a major peanut allergen, and a related conglutin ara h 6 were performed in Arachis hypogaea L. and its putative progenitors, Arachis duranensis and Arachis ipaensis. The complete identity between sequences encoding Ara h 2 isoforms demonstrated that these are homeologous genes and represent orthologs from diploid ancestors. Three copies of ara h 6 were identified in A. hypogaea, one of them located in the A-genome and the other two in the B-genome. Expression analysis showed higher levels of ara h 2 transcripts compared with ara h 6. Dual-labeled genomic in situ hybridization permitted identification of two subgenomes, each of which contained one pair of ara h 2-ara h 6 signals localized by fluorescence in situ hybridization. Characterization of genomic clones showed close genetic linkage between Ara h 2.02 and one copy of ara h 6 in the B-genome. The physical linkage may have arisen by tandem duplication and divergence of an ancestral gene. A gene duplication event specific to the B-genome progenitor has resulted in ara h 6 paralogs. These data provide further evidence for progenitor relationships and genomic organization of the conglutin gene family in the genus Arachis and could contribute to the development of a hypoallergenic peanut.

The Use of SNP Markers for Linkage Mapping in Diploid and Tetraploid Peanuts

Single nucleotide polymorphic markers (SNPs) are attractive for use in genetic mapping and marker-assisted breeding because they can be scored in parallel assays at favorable costs. However, scoring SNP markers in polyploid plants like the peanut is problematic because of interfering signal generated from the DNA bases that are homeologous to those being assayed. The present study used a previously constructed 1536 GoldenGate SNP assay developed using SNPs identified between two A. duranensis accessions. In this study, the performance of this assay was tested on two RIL mapping populations, one diploid (A. duranensis × A. stenosperma) and one tetraploid [A. hypogaea cv. Runner IAC 886 × synthetic tetraploid (A. ipaënsis × A. duranensis)4×]. The scoring was performed using the software GenomeStudio version 2011.1. For the diploid, polymorphic markers provided excellent genotyping scores with default software parameters. In the tetraploid, as expected, most of the polymorphic markers provided signal intensity plots that were distorted compared to diploid patterns and that were incorrectly scored using default parameters. However, these scorings were easily corrected using the GenomeStudio software. The degree of distortion was highly variable. Of the polymorphic markers, approximately 10% showed no distortion at all behaving as expected for single-dose markers, and another 30% showed low distortion and could be considered high-quality. The genotyped markers were incorporated into diploid and tetraploid genetic maps of Arachis and, in the latter case, were located almost entirely on A genome linkage groups.

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.

A Developmental Transcriptome Map for Allotetraploid Arachis hypogaea

The advent of the genome sequences of Arachis duranensis and Arachis ipaensis has ushered in a new era for peanut genomics. With the goal of producing a gene atlas for cultivated peanut (Arachis hypogaea), 22 different tissue types and ontogenies that represent the full development of peanut were sequenced, including a complete reproductive series from flower to peg elongation and peg tip immersion in the soil to fully mature seed. Using a genome-guided assembly pipeline, a homeolog-specific transcriptome assembly for Arachis hypogaea was assembled and its accuracy was validated. The assembly was used to annotate 21 developmental co-expression networks as tools for gene discovery. Using a set of 8816 putative homeologous gene pairs, homeolog expression bias was documented, and although bias was mostly balanced, there were striking differences in expression bias in a tissue-specific context. Over 9000 alterative splicing events and over 6000 non-coding RNAs were further identified and profiled in a developmental context. Together, this work represents a major new resource for cultivated peanut and will be integrated into peanutbase.org as an available resource for all peanut researchers.

Antifungal Activity in Transgenic Peanut (Arachis hypogaea L.) Conferred by a Nonheme Chloroperoxidase Gene

A nonheme chloroperoxidase gene (cpo-p )f rom Pseudomonas pyrrocinia, a growth inhibitor of mycotoxin-producing fungi, was introduced into peanut via particle bombardment. The expression of the cpo-p gene is predicted to increase pathogen defense in peanut. Embryogenic peanut tissues were bombarded with gold particles coated with plasmid pRT66 carrying the cpo-p and hygromycin phosphotransferase (hph) genes, under the control of a double CaMV 35S and a single CaMV 35S promoter, respectively. Selection for hygromycinresistant somatic embryos was performed on a liquid medium containing 10–20 mg/L hygromycin 3–4 days after bombardment. The integration and expression of the cpo-p gene was confirmed by Southern, Northern and Western blot analyses. In vitro bioassay using crude protein extracts from transgenic T0, T1, and T4 plants showed inhibition of Aspergillus flavus hyphal growth, which could translate to a reduction in aflatoxin contamination of peanut seed.

USE OF GREEN FLUORESCENT PROTEIN AS A NON-DESTRUCTIVE MARKER FOR PEANUT GENETIC TRANSFORMATION

SummaryThe ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.

Development and Phenotyping of Recombinant Inbred Line (RIL) Populations for Peanut (Arachis hypogaea)

ABSTRACT The identification of molecular markers for economically significant traits should greatly improve the speed and efficiency of all peanut (Arachis hypogaea L.) breeding programs. Developme...