Development of genomics resources for arachis hypogaea and profiling the genetic regulation of pre-harvest aflatoxin resistance
Clevenger, Josh Paul
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Aflatoxin is a highly carcinogenic mycotoxin produced by Aspergillus spp that contaminates peanuts (Arachis hypogaea) pre- and post-harvest. Understanding the interaction between peanut and fungus, leading to the production of aflatoxin, is key to identifying genetic sources of resistance. With the goal of describing the state of the seed that is permissive or repressive of pre-harvest aflatoxin contamination, a highly controlled, automated rainout shelter design was used to generate materials with differing conditions of Aspergillus colonization and aflatoxin contamination for RNA-sequencing of single seeds. To properly analyze these data new genomic and transcriptomic resources needed to be developed for peanut. First, 22 tissues and ontogenies were sampled that spanned the developmental stages of peanut and created a gene atlas. A genome-guided pipeline was used to create a homeolog-specific transcriptome assembly which was used to annotate 23 co-expression networks, over 9,000 alternative splicing events, and over 6,000 non-coding RNAs. Second, a novel pipeline, SWEEP, was developed for identifying high quality single nucleotide polymorphism (SNP) markers in peanut and was released as a freely available tool. Using SWEEP, a 60K SNP chip for Arachis spp was designed and used to investigate the genetic diversity of US peanut cultivars and to identify genomic regions that have undergone preferential selection by breeders. The developed transcriptome assembly and SNP filtering pipeline were used to analyze the RNA sequencing data from the rainout shelter experiment. Sets of genes were identified, across genotypes, which were differentially expressed between seeds contaminated with aflatoxin and not. Key pathways that were significantly differentially expressed were identified including fatty acid biosynthesis, GA biosynthesis, and ABA signaling. Additionally, potential breeding targets were found to increase pre-harvest resistance, including a repressor of ABA signaling, ABR1. SNPs associated with differentially expressed genes between genotypes were identified as potential eQTL. Finally, a set of non-coding RNAs were discovered that are differentially expressed, adding a layer of potential regulation for aflatoxin resistance. Overall, resource development facilitated the ability to describe the state of the seed that is permissive for aflatoxin contamination, which provides a platform for future pre-harvest aflatoxin contamination research.