Biotech and Potatoes: What does the future hold?

Using Plant Genomics to Meet the Needs of the 21st Century Dave Douches and C. Robin Buell
Michigan State University

Walter De Jong
Cornell University

How to Meet the Challenges of Crop Producton

The Green Revolution in the 20th century was due to the introduction of high yielding varieties using conventional breeding approaches coupled with the use of fertilizer and pesticides

One Few Traits at a time

Norman Borlag: 1970 Nobel Peace Price Winner: Using conventional breeding developed new strains of Wheat that were high yielding. This then was applied to rice and maize (corn)

A genome is the complete gene content of an organism

Understanding the potatos genetic map, or genome, will be a key component to genetic improvements of potato varieties For example, the Human Genome Project is finding the genes responsible for cancer, diseases, and other disorders For potato, we can conduct analogous research

Agricultural researchers have new tools to solve problems

Genomics is the study of whole genome of the organism Genomics is resulting in a major paradigm shift in biological research due to:
Having access to the entire gene complement of an organism rather than a few genes Scale at which experiments can be done

Genomics has the power to make a major impact in improvement of agriculture

Important Tool for Research: The Potato Genome Sequence

The Potato Genome Sequencing Consortium (PGSC) is an international group of scientists that have collaborated to sequence the genomes of two species potato: Solanum tuberosum and Solanum phureja.

What other genomes have been sequenced?

Potato 850 Mb Wheat: 16,000 Mb Rice: 430 Mb

5 Mb
Arabidopsis 130 Mb John Doe 2,500 Mb

Benefits of the Accessing the Potato Genome

The Potato Genome Sequencing Consortium is working to identify (annotate) all of the genes in the potato genome The sequencing of the potato genome will provide a major boost to gaining a better understanding of potato trait biology and will underpin future breeding efforts Access to these genes of the potato will allow the use of either conventional breeding methods or genetic engineering to try to create better potato varieties.

Annotation: to make or furnish critical or explanatory notes or comment. Merriam Websters Collegiate Dictionary -Annotation of genomes is driven by computers and human interpretation -Created new discipline of science: Bioinformatics = merge of computer science with biology

Further Benefits of the Accessing the Potato Genome

Overcome many negative aspects of potato as a genetic system (tetraploidy) using bioinformatics Facilitate gene isolation and allow molecular geneticists to accelerate trait gene discovery
Use a gene from tomato to locate a gene in potato

Radical effects on efficiency of breeding improved potato varieties with genetic markers (SNPs)
Enhance our ability to identify the desirable variants of genes underlying important traits such as starch, sugar, disease resistance, and nutrient content

What is a SNP?
Single-nucleotide polymorphism
(SNP, pronounced snip) SNP is a DNA sequence variation occurring when a single nucleotide A, T, C, or G in the genome differs between members of a species

Single-nucleotide polymorphisms may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence may or may not change the amino acid sequence of the protein that is produced.

Why does a breeder use genetic markers?

A genetic marker linked to a trait of interest, can be used to select indirectly for the marker rather than the trait. This is valuable when the trait is:
Multigenic (specific gravity) Hard to select for at early stages of breeding Expensive to screen for (nematodes, asn) Needed to be combined or pyramided (LB) Controlled by a recessive gene

Use of markers can make breeding more efficient and more effective.

For breeders to link markers to traits

Need to develop populations in which the trait is segregating so that it can be genetically mapped. Need enough genetic markers to make the process worthwhile. Needs to cost effective and time efficient Best if the marker is in the gene influencing the trait.

The challenge of mapping key alleles in economically relevant (elite) 4x potato germplasm
Number of genetic markers needed to construct a useful genetic map in tetraploid potato is more than twice number of markers needed for diploid potato How many SNPs are needed for routine mapping in 4x crosses? A function of desired marker density, and allele frequency. If want one simplex SNP every 10 cM 800 markers theory predicts we need 2000 SNPs or more.

SolCAP Primary Research Objective for

Potato
Developing SNP Markers in Elite Germplasm for Potato Breeding application Identify 10,000 SNPs in elite potato germplasm
The Illumina 10K potato chip was created from SolCAP SNPs

Use the SNPs to genotype with Illuminas Infinium platform, both diverse elite germplasm and bi-parental mapping populations Funding
This project is supported by the Agriculture and Food Research Initiative Applied Plant Genomics CAP Program of USDAs National Institute of Food and Agriculture

Using Bioinformatic SNPs were IDed in some key candidate genes

Sucrose-phosphate-synthase Soluble starch synthase 3, chloroplastic/amyloplastic Acid invertase Granule-bound starch synthase 2, chloroplastic/amyloplastic Glucose-6-phosphate isomerase Sucrose sythase Isoamylase isoform 2 Sucrose transporter Beta-amylase Sucrose synthase Granule-bound starch synthase 1 Phosphoglucomutase 20 18 16 10 10 10 8 8 6 6 6 6

With Bioinformatics we can develop an effective genome-wide set of SNP genomemarkers

Spacing and gene region coverage 2769 SNPs in candidate genes 508 SNPs in genetic markers 6723 SNPs from throughout the genome

How much of the genome is represented? ~650 Mb of the genome (of ~ 850 Mb genome)

SolCAP SNP Genotyping

Currently SolCAP is SNP genotyping 1,152 potato lines with 8,303 functioning SNPs (Potato SNP chip) potato germplasm panel: 325 clones 4x russet mapping population: 200 clones 2x mapping population: 94 clones Community SNP genotyping:
2 populations: 350

Primary benefit of the Potato SNP chip

Potato genotyping, especially at 4x level, is now simple and quick
isolate DNA send it to genotyping center receive a LOT of marker data in a week (a 3-day lab procedure)

a 4x cross for mapping

PairPair-wise Comparison of SNPs

W2310-3 x Kalkaska MSG227-2 x Jacqueline Lee Atlantic x Superior Stirling x 12601ad1 B1829-5 x Atlantic BER 63 x DM1-3 BER 83 x DM1-3 84SD22 x DM1-3 MCR205 x DM1-3 DI x DM1-3 08675-21 x 09901-01 RH x SH

4X 4X 4X 4X 4X 2X 2X 2X 2X 2X 2X 2X

22.4 16.5 5.9 25.9 11.5 79.3 78.8 46.0 76.7 85 53.8 59

37.6 51.8 51.8 37.6 18.8 20.7 21.2 54.0 23.3 15 46.2 41

40.0 31.8 42.4 36.5 69.8 0 0 0 0 0 0 0

zI = segregation not dependent on scoring dosage; II = segregation dependent on scoring dosage

What makes up the Potato Germplasm Panel Phenotypic Evaluation?

Clonal Study (CS)
250 clones 2 reps X 10 hills OR, WI, NY

Russet Mapping Population (MP)

Rio Grande X Premier Russet 200 progeny 2 reps X 10 hills ID, NC, MN

CS MP

MP CS

CS

MP

States in blue = Participants in SolCAP

Potato Germplasm Panel

Clonal Study (325 clones)
Top 50 N. American varieties Historical varieties Advanced breeding clones
from every US and Canadian program

Phenotypic evaluation
Key traits: specific gravity, sucrose, glucose, Vitamin C, maturity, tuber shape, tuber number, etc. Additional traits determined by breeding community Data curated at SGN

Non-American germplasm Genetic stocks

Analyses
Association mapping Parental selection Resolve population structure

Traits being evaluated within SolCAP

10 plant plots, two replicates
specific gravity chip color after cold storage sucrose/glucose

The key three

skin texture tuber shape (l/w/h) eye depth skin color, flower color flesh color
vine maturity (95, 120 dap) growth habit (prostrate, erect, etc) total yield heat sprouts internal defects

Traits being mapped in other populations

Chip color Reducing sugar Specific gravity Scab resistance Late blight resistance Vine maturity Asparagine concentration in tubers Acrylamide formation in chips More

SolCAP is creating databases for breeders

A database is being developed for the breeders to access the genetic information in alignment with the trait data being collected across multiple environments. The breeders will be able to more effectively map traits of interest and design breeding methods that will improve their ability to combine or enhance traits through selection

Do we stand a realistic chance of detecting useful marker-trait associations?

Where we are evaluating 220+ potato clones with 8000+ markers

Yes...

221 tetraploid potato clones x 250 AFLP markers >> 69 markers associated with 11 traits (P<0.01)
(chip color, after cooking darkening, maturity, shape...)

Whole-Genome Genotyping: The Infinium Assay

Infinium HD iSelect BeadChips iSelect is the custom chip Scalable from 10,000 SNPs per sample Unique oligo for each bead type Bead Pool is 250,000 bead types Random self-assembly of beads Average 15 to 30 beads of each type Three day lab procedure No gels in the lab (old school thinking)
http://www.illumina.com/technology/infinium_hd_assay.ilmn

12 and 24 sample HD (2 M) Infinium products

CytoSNP-12 PorcineSNP60 OvineSNP50 iSelect custom (60,801200,000+ attempted beadtypes) BovineSNP50 MaizeSNP50
iSelect custom (3,00060,800 attempted beadtypes)

BovineHD8 sample chip

28 28

Illuminas Core BeadArray Technology

29 29

Image

30 30

INTENSITY

INTENSITY

INTENSITY

Genome Studio Software

http://www.illumina.com/software/genomestudio_software.ilmn

Diploid Segregation

Black = Parents (AA x BB) Red = Population (all AB)

Black = Parents (AB x BB) Red = Population (1:1 AB:BB)

Genome Studio Software

Designed for use with diploid populations
Clusters are called as AA, AB, BB

Potato is tetraploid with at least 5 marker classes

AAAA, AAAB, AABB, ABBB, BBBB, also nulls (i.e. AAA)

http://www.illumina.com/software/genomestudio_software.ilmn

Good 5 Cluster Markers

Tetraploid Segregation

Yellow = Parents (AABB x BBBB) Red = Population (1:4:1 AABB:AAAB:BBBB)

Yellow = Parents (AABB x AABB) Red = Population (1:8:18:8:1 AAAA:AAAB:AABB:AAAB:BBBB)

SNP segregation in 4x Russet Mapping population (Premier x Rio Grande)

Cross Duplex x Duplex Duplex x Nulliplex Duplex x Simplex Nulliplex x Duplex Nulliplex x Nulliplex Nulliplex x Simplex Simplex x Duplex Simplex x Nulliplex Simplex x Simplex Simplex x Het Total

94 SNPs evaluated:
10 2 11 2 28 6 11 8 11 5 94 10 4 22 28 14

30% were Aaaa x aaaa 8% were AAaa x aaaa

11 5 94

Ideal Marker

Diploid mapping population Yellow Samples = Parents Red Samples = Population

Tetraploid mapping population Yellow Samples = Parents Red Samples = Population

This marker is ideal of both 2x and 4x germplasm. The 2x AA cluster overlaps the 4x AAAA cluster, the 2x AB cluster overlaps the 4x AABB cluster, and the 2x BB cluster overlaps the 4x BBBB cluster.

Next Steps in 2011

What do we do with all the marker data collected? MAP QTL Training of breeders to use SNP technology

Outcomes for Breeding from SolCAP

A genome-wide set of markers genomeand bioinformatic tools accessible by breeders
Breeders will access germplasm for crossing based upon SNP polymorphism and linked QTL of interest Design crosses complementary for QTL and traits, and then use marker assisted breeding.

Outcomes for Breeding from SolCAP

Better understanding of the allelic variation influencing carbohydrates
Design crosses to create improved sugar and starch levels and starch quality. Crosses to manipulate and select variation within existing elite populations or introgress novel alleles from wild germplasm. More predictable and directed breeding effort for processing and fresh market traits.

Visit us at http://solcap.msu.edu/