Application of molecular marker technologies on cereal crops

improvement

Shiaoman Chao

USDA-ARS, Fargo, ND

WA

KSNC

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

Breeder Location

Wheat (HRS)James Anderson MinnesotaElias Elias (durum) North DakotaKarl Glover South DakotaMohamed Mergoum North Dakota

Barley (Spring 6 rowed)Kevin Smith Minnesota

Genotyping Project Work Flow

genomic DNA extraction

PCR reactions

gel electrophoresis

data retrieval

Genotyping Lab Breeders

sample preparationand 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 extraction2. PCR reaction setup3. Gel electrophoresis4. 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 markerDNA extraction $0.12PCR reactions $0.06Gel 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 markerFP assay $0.60Total 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

InitiativeUSDA CSREES wheat and barley CAP

fundsUSDA-ARS CRIS funds