Location: National Clonal Germplasm Repository for Citrus
Project Number: 2036-21000-012-013-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2025
End Date: Aug 31, 2026
Objective:
Provides expertise in the area of genetic analysis of citrus and date germplasm. This supports the mission of the Repository to collect, maintain, characterize, evaluate, distribute , and document germplasm of citrus, date palms, and related taxa. Specific objectives of this agreement are: (1) assess genomic changes that may have occurred during cryopreservation of citrus germplasm; (2) screen germplasm accessions for ploidy level; (3) assess the embryony level of germplasm accessions and the percentage of zygotic embryos in polyembryonic accessions.
Approach:
(1) To assess genomic changes that may have occurred during cryopreservation, whole genome re-sequencing of regenerated accession will be carried out using an Illumina® NovaSeq 6000 S4 platform. Young leaf tissue will be sampled from regenerated trees and the source trees from which the materials were cryopreserved. Sequencing libraries will be prepared and sequenced (150 bp paired end reads) with a target depth of at least at 100X. Variant sites will be mined using a variant discovery pipeline using Phytozome C. clementina v1.0 reference genome for nuclear genome variant discovery. The paired end reads will be mapped to the haploid clementine reference sequence using bwa/mem, followed by removal of duplicated reads using Picard. The Genome Analysis Toolkit (GATK) HaplotypeCaller will be used for Single Nucleotide Polymorphism (SNP) calls. (2) Ploidy analysis will be assessed using nuclei isolated from ~30-50 mg of young leaf tissue collected from two trees of each accession (two trees being available for the majority of accessions). Three biological replicates per individual will be collected and analyzed. Flow cytometry analysis will be conducted using the NovoCyte flow cytometer system located at the UCR School of Medicine. (3) In order to assess the embryony level, 100 seeds will be germinated from each accession studied. The number of seedlings produced will indicated whether the accession is mono- or poly-embryonic. The percentage zygotic seedlings will be determined using KASP (Kompetitive Allele Specific PCR) SNP markers (6 to 16) that were designed by LGC Genomics from 50 bp of the Clementine reference genome sequence flanking each SNP.