Application of molecular marker technologies on cereal crops improvement Shiaoman Chao USDA-ARS, Fargo, ND

WA KS NC EAST NORTH CENTRAL CENTRAL WEST USDA-ARS Small Grains Regional Genotyping Centers ND

Exploitation of High Throughput Marker Systems  SSRs  SNPs - detection system Fluorescent Polarization (FP), a single SNP assay system, suitable for genotyping a small number of SNPs on a large number of samples, such as MAS. Illumina’s BeadArray, a high throughput platform for 1,536-plex SNP GoldenGate assay, suitable for whole genome scan and association mapping studies  DArT - Diversity Array Technology, an arrayed based marker system, developed by Andrzej Kilian at Canberra in Australia. (Cost: US$3,500/96 samples)

Marker-Assisted Breeding Programs BreederLocation Wheat (HRS) James AndersonMinnesota Elias Elias (durum)North Dakota Karl GloverSouth Dakota Mohamed MergoumNorth Dakota Barley (Spring 6 rowed) Kevin SmithMinnesota

Genotyping Project Work Flow genomic DNA extraction PCR reactions gel electrophoresis data retrieval Genotyping LabBreeders sample preparation and submission data analysis 2 months turnaround time

Sample Submission Protocol from Breeders  96-deep-well plates (2ml) are provided  Add ~0.25 gm of silica gel (16-20 mesh) into each well  Cut and insert one piece of 11/2’’ leaf fragment in each well, dry samples at room temp  Seal plates and mail plates to Fargo  Store plates at room temp

High Throughput Genotyping Protocol Liquid handling is automated. 1.Plant DNA extraction 2.PCR reaction setup 3.Gel electrophoresis 4.Data analysis – allele calls is done using a software

Current Projects on MAS - Sample Processing Approximate current capacity:  DNA extraction - process 12 to 16 plates per day  Generate 8,000 data points/week  A maximum 30,000-data point project (or 8,000 samples genotyped with up to 4 markers) is expected to take ~2 months from sample submission to data delivery

Current Marker-Assisted Breeding Projects - collaborative projects with breeders in the public sector through grants  wheat and barley MAS projects for FHB resistance.  Marker validation associated with FHB resistance derived from various sources in wheat.  MAS projects from wheat CAP

Cost per Genotype SSR marker DNA extraction$0.12 PCR reactions$0.06 Gel electrophoresis$0.12 (4-marker assay) $0.50 (1-marker assay) Labor$0.30 Total cost$0.60 for 4-marker assay $1.00 for single marker assay SNP marker FP assay$0.60 Total cost$1.10 Illumina’s system<$0.05 (consumables)

Effectiveness and Cost Efficiency of MAS  Improving marker technologies, developing diagnostic markers.  Breeding strategies backcrossing vs. two-way or topcrossing selection strategies, allele enrichment vs. fixation, population size number of marker/trait to be screened

Fargo Genotyping Lab Staff Lee Brady (Cat 3) - coordinate and perform MAS projects Jamie Rust (Technician) - perform research projects and assist MAS projects Term appointment (Technician) - SNP development using Illumina’s technology and assist MAS projects

Acknowledgements Funding sources, USDA-ARS/US Wheat and Barley Scab Initiative USDA CSREES wheat and barley CAP funds USDA-ARS CRIS funds