Application of molecular marker technologies on cereal crops

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

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

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 Bead. Array, a high throughput platform for 1, 536 -plex SNP Golden. Gate assay, suitable for whole genome scan and association mapping studies § DAr. T - 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 Breeder Location Wheat (HRS) James Anderson Elias (durum) Karl Glover Mohamed Mergoum Minnesota North Dakota South Dakota North Dakota Barley (Spring 6 rowed) Kevin Smith Minnesota

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

Sample Submission Protocol from Breeders § 96 -deep-well plates (2 ml) 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 PCR reactions Gel electrophoresis $0. 12 $0. 06 $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 Total cost $0. 60 $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