Location: Sustainable Perennial Crops Laboratory
Project Number: 8042-21000-303-013-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Feb 26, 2025
To develop novel SNP markers and other genomic tools for molecular characterization of genetic resources of guava, rambutan, pitaya, star fruit, mangosteen, tea and peach palm.
For the development of SNP markers, young leaf tissues will be collected from 8-10 varieties maintained in the collections in Hilo, Hawaii, Mayaguez, Puerto Rico or SPCL's greenhouse in Beltsville, MD. The selection of varieties will be based on their geographical origins. Leaf tissues will be sampled, and total RNA will be extracted for each leaf tissue separately using the RNeasy plant mini kit (Qiagen, Hilden, Germany). For each variety, the RNA extracted from three leaves will be evenly pooled. RNA purity and integrity will be confirmed using NanoDrop, Agilent 2100 Bioanalyzer and gel electrophoresis. The total RNA samples will then be sent to Beijing Genomics Institute BGI (China) for RNA-seq library preparation and sequencing and bioinformatics analysis. Putative SNP markers will be identified by SNP callers that fulfill the thresholds of read depth and allele frequency. The identified SNPs then will be screened using Fluidigm nanofluidic genotyping system. The USDA-ARS NPGS gene-bank collections of tropical fruits and nuts germplasm accessions in Hilo, Hawaii and Mayaguez, Puerto Rico will be genotyped and high-quality SNPs identified. To form a genotyping panel for each species, polymorphisms, heterozygosity, minor allele frequency, and repeatability criteria will be used. PCR amplification and SNP analysis will follow standard Fluidigm protocols for the Fluidigm EP1 Dynamic Array IFC (integrated fluidic circuit) system. This array processes 96 samples with 96 SNP markers resulting in 9,216 data points from each array. For the development of genetic linkage maps, genotyping-by-Sequencing (GBS) will be performed on mapping populations of these species. Genotyping service will be provided by LGC Genomics, Ltd which has secured a license for the commercial offering and use of GBS technology from KeyGene. Quality filtering of genetic markers will be performed to remove SNPs with greater than 10% missing data by locus. In addition, a minimum allele frequency of 5% will be applied to reduce the impact of false polymorphism. The retained SNPs will be used for constructing genetic maps for these species. Genetic mapping will be performed using JoinMap® 4.1. Marker groupings will be manually verified by inspecting the grouped markers for agreement with known physical locations. Marker order and distances will be calculated using the Maximum Likelihood Mapping function with default settings. Segregation distortion of markers will be calculated using JoinMap.