Location: Sugarcane Research
Project Number: 6052-21000-018-011-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2023
End Date: Jul 31, 2026
Objective:
A medium-density SNP array will be developed using SNPs which are evenly distributed throughout the sugarcane genome. These SNPs were previously mapped using a monoploid genome of sugarcane as well as a genome of Saccharum spontaneum. The genomic variants will be carefully selected so they represent different accessions and traits that can be tracked in the basic breeding progenies and subsequently in commercial sugarcane hybrids.
Approach:
The PIs have completed genotyping of 458 clones including wild accessions, early-generation hybrids from crosses involving wild accessions, and commercial/elite sugarcane hybrids. More than 60,000 high confidence genomic variants have been identified. The number of SNPs will be further reduced based on: (1) selection of bi-allelic SNPs; (2) SNPs with minor allele frequency more than 0.1; and 3) exclusion of SNPs with missing calls in >20% genotypes. Linkage disequilibrium (LD) will be calculated. Haplotype blocks will be identified representing groups of SNPs with alleles that are co-inherited due to linkage, and the minimum number of SNP markers needed to uniquely identify each haplotype block will be selected. Oligos will be designed flanking ~10,000 selected SNPs representing LD blocks in the genome using the proprietary software of a commercial facility that has established research partnership with the research team. The selected SNPs will include some that are associated with desirable traits and unique to wild accessions. PCR amplification will be conducted with all wild accessions and a random subset of commercial/elite clones. The barcoded amplicon libraries will be sequenced using an NGS platform and allele frequency-based genotype calling will be performed using proprietary software. These allele calls will be tallied with genotype calls previously established from sequencing by the PIs for validation of their reproducibility. The validated SNPs (~8,000; ca. 80% validity) will form the panel to study the genetic diversity of the wild accessions.
Leaf tissues will be collected from all the wild accessions and DNA will be isolated using CTAB miniprep method. Genotyping will be performed using the SNP panel at the commercial facility. Genotyping data will be used to determine the genetic (dis)similarity among the accessions. Accession-specific alleles will be tracked in the basic germplasm and advanced clones using the SNP panel. In addition, the SNP panel will be used for other molecular breeding applications such as marker-assisted backcrossing, hybrid validation in basic germplasm, QTL mapping, trait profiling, and genomic selection. The SNP panel will be augmented with additional (or substituted) SNP markers as new accessions are introduced into the sugarcane germplasm enhancement program and/or when new SNPs associated with traits of interest are identified. Such changes in SNP composition are required for QA applications as the diversity of germplasm changes.
