application of molecular marker technologies on cereal crops improvement shiaoman chao usda-ars,...
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Application of molecular marker technologies on cereal crops
improvement
Shiaoman Chao
USDA-ARS, Fargo, 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
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