Josh Clevenger

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.

Development and Evaluation of a High Density Genotyping 'Axiom_Arachis' Array with 58 K SNPs for Accelerating Genetics and Breeding in Groundnut.

Single nucleotide polymorphisms (SNPs) are the most abundant DNA sequence variation in the genomes which can be used to associate genotypic variation to the phenotype. Therefore, availability of a high-density SNP array with uniform genome coverage can advance genetic studies and breeding applications. Here we report the development of a high-density SNP array ‘Axiom_Arachis’ with 58 K SNPs and its utility in groundnut genetic diversity study. In this context, from a total of 163,782 SNPs derived from DNA resequencing and RNA-sequencing of 41 groundnut accessions and wild diploid ancestors, a total of 58,233 unique and informative SNPs were selected for developing the array. In addition to cultivated groundnuts (Arachis hypogaea), fair representation was kept for other diploids (A. duranensis, A. stenosperma, A. cardenasii, A. magna and A. batizocoi). Genotyping of the groundnut ‘Reference Set’ containing 300 genotypes identified 44,424 polymorphic SNPs and genetic diversity analysis provided in-depth insights into the genetic architecture of this material. The availability of the high-density SNP array ‘Axiom_Arachis’ with 58 K SNPs will accelerate the process of high resolution trait genetics and molecular breeding in cultivated groundnut.

Tetrasomic Recombination Is Surprisingly Frequent in Allotetraploid Arachis

Arachis hypogaea L. (cultivated peanut) is an allotetraploid (2n = 4x = 40) with an AABB genome type. Based on cytogenetic studies it has been assumed that peanut and wild-derived induced AABB allotetraploids have classic allotetraploid genetic behavior with diploid-like disomic recombination only between homologous chromosomes, at the exclusion of recombination between homeologous chromosomes. Using this assumption, numerous linkage map and quantitative trait loci studies have been carried out. Here, with a systematic analysis of genotyping and gene expression data, we show that this assumption is not entirely valid. In fact, autotetraploid-like tetrasomic recombination is surprisingly frequent in recombinant inbred lines generated from a cross of cultivated peanut and an induced allotetraploid derived from peanut’s most probable ancestral species. We suggest that a better, more predictive genetic model for peanut is that of a “segmental allotetraploid” with partly disomic, partly tetrasomic genetic behavior. This intermediate genetic behavior has probably had a previously overseen, but significant, impact on the genome and genetics of cultivated peanut.

SWEEP: A Tool for Filtering High-Quality SNPs in Polyploid Crops

High-throughput next-generation sequence-based genotyping and single nucleotide polymorphism (SNP) detection opens the door for emerging genomics-based breeding strategies such as genome-wide association analysis and genomic selection. In polyploids, SNP detection is confounded by a highly similar homeologous sequence where a polymorphism between subgenomes must be differentiated from a SNP. We have developed and implemented a novel tool called SWEEP: Sliding Window Extraction of Explicit Polymorphisms. SWEEP uses subgenome polymorphism haplotypes as contrast to identify true SNPs between genotypes. The tool is a single command script that calls a series of modules based on user-defined options and takes sorted/indexed bam files or vcf files as input. Filtering options are highly flexible and include filtering based on sequence depth, alternate allele ratio, and SNP quality on top of the SWEEP filtering procedure. Using real and simulated data we show that SWEEP outperforms current SNP filtering methods for polyploids. SWEEP can be used for high-quality SNP discovery in polyploid crops.

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.

Gene expression profiling describes the genetic regulation of Meloidogyne arenaria resistance in Arachis hypogaea and reveals a candidate gene for resistance

Resistance to root-knot nematode was introgressed into cultivated peanut Arachis hypogaea from a wild peanut relative, A. cardenasii and previously mapped to chromosome A09. The highly resistant recombinant inbred RIL 46 and moderately resistant RIL 48 were selected from a population with cv. Gregory (susceptible) and Tifguard (resistant) as female and male parents, respectively. RNA-seq analysis was performed on these four genotypes using root tissue harvested from root-knot nematode infected plants at 0, 3, 7 days after inoculation. Differential gene expression analysis provides evidence that root-knot nematodes modulate biological pathways involved in plant hormone, defense, cell signaling, cytoskeleton and cell wall metabolism in a susceptible reaction. Corresponding to resistance reaction, an effector-induced-immune response mediated by an R-gene was identified in Tifguard. Mapping of the introgressed region indicated that 92% of linkage group A09 was of A. cardenasii origin in Tifguard. RIL46 and RIL 48 possessed 3.6% and 83.5% of the introgression on A09, respectively. Within the small introgressed region carried by RIL 46, a constitutively expressed TIR-NBS-LRR gene was identified as the candidate for nematode resistance. Potential defense responsive pathways include effector endocytosis through clathrin-coated vesicle trafficking, defense signaling through membrane lipid metabolism and mucilage production.

Identification of Rare Recombinants Leads to Tightly Linked Markers for Nematode Resistance in Peanut

ABSTRACT Strong host resistance to root-knot nematode (RKN; Meloidogyne arenaria) introgressed from a wild diploid species to cultivated peanut was previously shown to be located on a large chromos...

Genotypic Regulation of Aflatoxin Accumulation but Not Aspergillus Fungal Growth upon Post-Harvest Infection of Peanut (Arachis hypogaea L.) Seeds

Aflatoxin contamination is a major economic and food safety concern for the peanut industry that largely could be mitigated by genetic resistance. To screen peanut for aflatoxin resistance, ten genotypes were infected with a green fluorescent protein (GFP)—expressing Aspergillus flavus strain. Percentages of fungal infected area and fungal GFP signal intensity were documented by visual ratings every 8 h for 72 h after inoculation. Significant genotypic differences in fungal growth rates were documented by repeated measures and area under the disease progress curve (AUDPC) analyses. SICIA (Seed Infection Coverage and Intensity Analyzer), an image processing software, was developed to digitize fungal GFP signals. Data from SICIA image analysis confirmed visual rating results validating its utility for quantifying fungal growth. Among the tested peanut genotypes, NC 3033 and GT-C20 supported the lowest and highest fungal growth on the surface of peanut seeds, respectively. Although differential fungal growth was observed on the surface of peanut seeds, total fungal growth in the seeds was not significantly different across genotypes based on a fluorometric GFP assay. Significant differences in aflatoxin B levels were detected across peanut genotypes. ICG 1471 had the lowest aflatoxin level whereas Florida-07 had the highest. Two-year aflatoxin tests under simulated late-season drought also showed that ICG 1471 had reduced aflatoxin production under pre-harvest field conditions. These results suggest that all peanut genotypes support A. flavus fungal growth yet differentially influence aflatoxin production.

RNA Sequencing of Contaminated Seeds Reveals the State of the Seed Permissive for Pre-Harvest Aflatoxin Contamination and Points to a Potential Susceptibility Factor

Pre-harvest aflatoxin contamination (PAC) is a major problem facing peanut production worldwide. Produced by the ubiquitous soil fungus, Aspergillus flavus, aflatoxin is the most naturally occurring known carcinogen. The interaction between fungus and host resulting in PAC is complex, and breeding for PAC resistance has been slow. It has been shown that aflatoxin production can be induced by applying drought stress as peanut seeds mature. We have implemented an automated rainout shelter that controls temperature and moisture in the root and peg zone to induce aflatoxin production. Using polymerase chain reaction (PCR) and high performance liquid chromatography (HPLC), seeds meeting the following conditions were selected: infected with Aspergillus flavus and contaminated with aflatoxin; and not contaminated with aflatoxin. RNA sequencing analysis revealed groups of genes that describe the transcriptional state of contaminated vs. uncontaminated seed. These data suggest that fatty acid biosynthesis and abscisic acid (ABA) signaling are altered in contaminated seeds and point to a potential susceptibility factor, ABR1, as a repressor of ABA signaling that may play a role in permitting PAC.