Location: Crop Improvement and Genetics Research
Project Number: 2030-21430-014-007-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Mar 1, 2018
End Date: Feb 28, 2023
Objective:
1. Use reduced representation sequencing approaches to genotype a switchgrass population displaying heterosis for yield.
2. Quantify gene expression levels in pooled individuals that differ most in yield.
Approach:
We will use targeted gene capture with predesigned bait libraries and biotinylated RNA-based probes (baits) with a length of 120 nucleotides (nt) that correspond to annotated exon sequences in switchgrass. Overlapping baits will allow for a 60 nt overlap to neighboring baits to facilitate 2x coverage. For hybridization, genomic DNA samples will be prepared from the switchgrass population. The genomic DNA will be sheared to obtain an average fragment size of approximately 800 bp and hybridized with the customized bait library for targeted-sequence enrichment. Enriched DNA from individual samples will be used to prepare a sequencing library with a unique barcode for Illumina sequencing. After sequencing, reads will be extracted for each sample based on the unique barcodes and used to map to the reference sequences. Bioinformatics data analysis will identify any sequence differences among different switchgrass lines.
For RNA sequencing, we will create sequencing pools by separation of high and low yielding individuals. Total RNA will be isolated from these individuals and reverse transcribed into cDNA using the SMARTer PCR cDNA Synthesis Kit that has been optimized for preparing high-quality, full-length cDNA. Illumina sequencing libraries for each pool and biological replicate will be constructed using unique barcode adaptors and sequenced on the Illumina HiSeq4000 platform.
