biotech and potatoes what does the future hold

8/2/2019 Biotech and Potatoes What Does the Future Hold

1/21

Biotech and Potatoes: What

does the future hold?

Dave Douches and C. Robin BuellMichigan State University

Walter De Jong

Cornell University

Using Plant Genomics to Meet the Needs of

the 21stCentury

How to Meet the Challenges of Crop Producton

The Green Revolution in the 20th century was due tothe introduction of high yielding varieties using

conventional breeding approaches coupled with the

use of fertilizer and pesticides

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)

One Few

Traits at a time


2/21

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


3/21

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: Solanumtuberosum and Solanum phureja.

Wheat:

16,000 Mb

Arabidopsis

130 Mb

John Doe

2,500 Mb

5 Mb

Rice:

430 Mb

What other genomes havebeen sequenced?

Potato

850 Mb


4/21

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 tocreate 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


5/21

Further Benefits of the

Accessing the Potato Genome

Overcome many negative aspects of potato as agenetic 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 ofgenes underlying important traits such as starch, sugar,

disease resistance, and nutrient content

Single-nucleotide polymorphism(SNP, pronounced snip)

SNP is a DNA sequence variation occurring when

a single nucleotide A, T, C, or G in thegenome 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.

What is a SNP?What is a SNP?


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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 geneinfluencing the trait.


7/21

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 mappingHow many SNPs are needed for routine mapping

in 4x crosses?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.

SolCAPSolCAP Primary Research Objective forPrimary Research Objective forPotatoPotato

Developing SNP Markers in Elite Germplasm for PotatoBreeding 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

FundingThis project is supported by the Agriculture and Food

Research Initiative Applied Plant Genomics CAP

Program of USDAs National Institute of Food and

Agriculture


8/21

Using Bioinformatic SNPs wereUsing Bioinformatic SNPs were IDedIDed inin

some key candidate genessome key candidate genes

Sucrose-phosphate-synthase 20

Soluble starch synthase 3, chloroplastic/amyloplastic 18

Acid invertase 16

Granule-bound starch synthase 2,

chloroplastic/amyloplastic 10

Glucose-6-phosphate isomerase 10

Sucrose sythase 10

Isoamylase isoform 2 8

Sucrose transporter 8

Beta-amylase 6

Sucrose synthase 6

Granule-bound starch synthase 1 6

Phosphoglucomutase 6

With Bioinformatics we can develop anWith Bioinformatics we can develop an

effective genomeeffective genome--wide set of SNPwide set of SNPmarkersmarkers

SpacingSpacing and gene region coverageand 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)


9/21

SolCAP SNP GenotypingSolCAP SNP Genotyping

Currently SolCAP is SNP genotyping 1,152 potatolines with 8,303 functioning SNPs (Potato SNPchip)

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 chipPrimary 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


10/21

PairPair--wise Comparison of SNPswise Comparison of SNPs

W2310-3 x Kalkaska 4X 22.4 37.6 40.0

MSG227-2 x Jacqueline Lee 4X 16.5 51.8 31.8

Atlantic x Superior 4X 5.9 51.8 42.4

Stirling x 12601ad1 4X 25.9 37.6 36.5

B1829-5 x Atlantic 4X 11.5 18.8 69.8

BER 63 x DM1-3 2X 79.3 20.7 0

BER 83 x DM1-3 2X 78.8 21.2 0

84SD22 x DM1-3 2X 46.0 54.0 0

MCR205 x DM1-3 2X 76.7 23.3 0

DI x DM1-3 2X 85 15 0

08675-21 x 09901-01 2X 53.8 46.2 0

RH x SH 2X 59 41 0

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

scoring dosage

What makes up the PotatoWhat makes up the Potato GermplasmGermplasm PanelPanel

Phenotypic Evaluation?Phenotypic Evaluation?

ClonalClonal Study (CS)Study (CS)

250 clones

2 reps X 10 hills

OR, WI, NY

Russet Mapping Population (MP)Russet Mapping Population (MP)

Rio Grande X Premier Russet

200 progeny

2 reps X 10 hills

ID, NC, MN

CSCS CS

MP

MP

MP

States in blue = Participants in SolCAP


11/21

Potato Germplasm PanelPotato Germplasm Panel

ClonalClonal Study (325 clones)Study (325 clones) Top 50 N. American varieties

Historical varieties

Advanced breeding clones

from every US and Canadian

program

Non-American germplasm

Genetic stocks

AnalysesAnalyses

Association mapping

Parental selection

Resolve population structure

Phenotypic evaluationPhenotypic evaluation Key traits: specific gravity,

sucrose, glucose, Vitamin C,maturity, tuber shape, tuber

number, etc.

Additional traits determined bybreeding community

Data curated at SGN

Traits being evaluated within SolCAP10 plant plots, two replicates

specific gravity chip color after cold storage sucrose/glucose

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

The key three


12/21

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 designbreeding methods that will improve their

ability to combine or enhance traits

through selection


13/21

Do we stand a realistic chance

of detecting useful marker-trait

associations?

Where we are evaluating 220+

potato clones with 8000+ markers

221 tetraploid potato clones x 250 AFLP markers

>> 69 markers associated with 11 traits (P


14/21

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

2828

12 and 24 sample HD (2 M) Infinium products

CytoSNP-12

PorcineSNP60

OvineSNP50

iSelect custom(60,801-

200,000+

attempted

beadtypes)

BovineSNP50

MaizeSNP50

iSelect custom (3,000-

60,800 attempted

beadtypes)

BovineHD8 sample chip


15/21

2929

Illuminas Core BeadArray Technology

3030

Image

C C G A TT

INTENSITY

INTENSITY

INTENSITY


16/21

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)


17/21

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


18/21

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)

Cross

Duplex x Duplex 10 10

Duplex x Nulliplex 2 4

Duplex x Simplex 11 22

Nulliplex x Duplex 2

Nulliplex x Nulliplex 28 28

Nulliplex x Simplex 6 14

Simplex x Duplex 11

Simplex x Nulliplex 8

Simplex x Simplex 11 11

Simplex x Het 5 5

Total 94 94

SNP segregation in 4x RussetSNP segregation in 4x Russet

Mapping population (Premier x Rio Grande)Mapping population (Premier x Rio Grande)

94 SNPs evaluated:

30% were Aaaa x aaaa

8% were AAaa x aaaa


19/21

Ideal Marker

Diploid mapping population

Yellow Samples = ParentsRed Samples = Population

Tetraploid mapping population

Yellow Samples = ParentsRed 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


20/21

Outcomes for Breeding fromOutcomes for Breeding from SolCAPSolCAP

A genomeA genome--wide set of markerswide set of markersandand bioinformaticbioinformatic toolstoolsaccessible by breedersaccessible by breeders

Breeders will access germplasmfor crossing based upon SNPpolymorphism and linked QTL ofinterest

Design crosses complementary for

QTL and traits, and then usemarker assisted breeding.

Outcomes for Breeding fromOutcomes for Breeding from SolCAPSolCAP Better understanding of theBetter understanding of the

allelic variation influencingallelic variation influencingcarbohydratescarbohydrates

Design crosses to createimproved sugar and starchlevels and starch quality.

Crosses to manipulate andselect variation within existingelite populations or introgress

novel alleles from wildgermplasm.

More predictable and directedbreeding effort for processingand fresh market traits.


21/21

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

biotech and potatoes what does the future hold

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