Application of molecular marker technologies on cereal crops improvement

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

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

3. 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)

4. Marker-Assisted Breeding Programs Breeder Location Wheat (HRS) James Anderson Minnesota Elias Elias (durum) North Dakota Karl Glover South Dakota Mohamed Mergoum North Dakota Barley (Spring 6 rowed) Kevin Smith Minnesota

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

6. 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

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

8. 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

9. 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

10. 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)

11. 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

12. 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

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