AN APPLE A DAY: INTERROGATING APPLE FOR CROP NUTRITIONAL QUALITY IMPROVEMENT

Our long-term goal is to produce unique apples that have enhanced health benefits, worthy of government-sanctioned health claims, that provide value to farmers, processors, and consumers. We propose the following specific objectives.Objective 1: Screen diverse apple germplasm to understand variation in chlorogenic acid and other phenolics across cultivated and wild apples.Apples will be sampled across an array including the most common commercially grown varieties, up-and-coming selections, progeny populations and their associated pedigree, and wild material. Environmental replication will be included for a subset of apples.A quantitative liquid-liquid extraction and liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis method will be developed and applied to fruits for chlorogenic acid and other common phenolics (e.g., flavanols, flavan-3-ols, anthocyanins, procyanidins, dihydrochalcones, and phenolic acids) in apples for which authentic standards are available. This approach will allow both a focused screening of chlorogenic acid and a broader understanding of phenolic distribution and profile among the selected germplasm.The outcomes of this objective will enable selection of the germplasm to be screened genomically and transcriptomically (see Objective 2 for details).Objective 2: Refine knowledge of genetic control of chlorogenic acid and phenolics in apple fruit by combining whole genome sequencing, transcriptomics, and phytochemical quantitative analysis.Germplasm across the chlorogenic acid and phenolic content range will be subjected to 1) long-read PacBio sequencing (n=10 most divergent genotypes), 2) gene expression analysis via Illumina RNA sequencing (top 5 five cultivars replicated over at least three environments), and 3) quantitative metabolite analysis using LC-MS/MS.This information will be used in order to refine genomic polymorphism-phenolics associations we have demonstrated previouslyon chromosomes 17 (and possibly 3). This will focus our understanding of the genetic control of chlorogenic acid and phenolics content in apple fruit at the transcript level.The outcome of this objective will be a set of haplotypes associated with chlorogenic acid/phenolic compounds across a diversity of germplasm that includes commercial cultivars, selections, and segregating populations.Objective 3: Understanding inheritance and genetic components involved in chlorogenic acid and phenolic content in apple fruits.We will evaluate germplasm generated through rapid cycle, fast breeding, and accelerated introgression with reduced juvenility. The material under current development has potential horticultural value (i.e., genetic background from commercially proven cultivars) with ~1800 seedlings already produced.Based on the findings in objectives 1 and 2, we will select existing seedlings, in addition to creating new reduced juvenility material. This will enable the production of fruit in less than two years (as opposed to the usual 6-12 years) in several small segregating families.Development of divergent apple germplasm for chlorogenic acid and phenolics is in progress, and additional progeny will be produced to contrast gene expression in bulk-segregantand inheritance analysesto identify genetic components (i.e., alleles) governing phytochemical production.To advance in allele identification and transcript association we will use RNA-sequencing using SMRT sequencing through PacBio Iso-seq of the most divergent segregating germplasm.The outcome of this objective will allow development of DNA tests that will enable the selection of desirable germplasm for future basic studies, clinical studies with animal models and human trials, as well as for future breeding endeavors.