Location: Southern Horticultural Research Unit
Project Number: 6062-21000-011-001-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 30, 2020
End Date: Aug 29, 2024
Objective:
1. Generate high-quality genome assembly of muscadine grape using Illumina, PacBio, and Dovetail Chicago or BioNano technologies.
2. Develop a high-throughput genotyping platform to locate genomic regions associated with phenological and fruit quality traits.
3. Conduct comparative transcriptome analysis to identify candidate genes potentially involved in resistance to X. fastidiosa.
Approach:
The PacBio sequence data will be assembled using either Dovetail or BioNano technologies to generate a high-quality genome assembly of muscadine grape. Eight diverse muscadine cultivars will be sequenced using Illumina platform. Reads will be processed, map to the draft genome and variants will be called. Initially we will select single nucleotide polymorphism (SNPs) within genes and then add non-genic SNPs as needed to ensure one region per 100 kb. A total of 5000 regions will be used for primers design. To achieve high level of multiplexing and increase precision of target sequence amplification, rhAmpSeq amplicon sequencing system developed by integrated DNA technologies will be used to genotype parents and individuals of the five mapping populations. To better capture linkage disequilibrium (LD) between SNPs, the entire amplicon of 200-300 bp will be used as haplotype allele tag instead of multiple independent SNPs separately. Further, 2000 rhAmpSeq markers developed by USDA ARS, grape genetics research unit, Geneva, New York to target only the core Vitis genome (Zou et al. 2020) will be used to genotype DNA from the five mapping populations.
Parents and individuals of the mapping populations will be characterized for dates of 50% bloom (DB), dates of 50% ripening (DR), fruit development period (FDB), resistance to X. fastidiosa. Berry weight, berry size, berry firmness, total soluble solids (TSS), total titratable acidity (TTA) and storage capability. Also, flavonoids (myricetin, quercetin, kaempferol, and anthocyanins) will be determined in parents and individuals of the mapping populations using HPLC. To define genomic regions associated with traits of interest and estimate the effects of their alleles, resulting genotypic data will be used to generate high-resolution genetic map for each population and integrated with phenotypic data to conduct QTL analyses.
The RNA-Seq data will be mapped against the muscadine draft genome. After alignment, the relative abundance of transcripts will be measured as reads per kilobase of exon model (RPKM). The differential expression between inoculated and non-inoculated plants and its significance will be calculated. The differentially expressed transcripts will be annotated, categorized using gene ontology, and functionally analyzed. Candidate genes identified by RNA-Seq will be further validated by RT-PCR. The most valuable outcomes of this project will be a genotyping platform that muscadine breeders can use to improve genetic gain, increase breeding efficiency, and estimate genetic diversity of muscadine grapes germplasm collection.
