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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
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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
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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
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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
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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.
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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
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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)
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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
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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
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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
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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
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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
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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
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2929
Illuminas Core BeadArray Technology
3030
Image
C C G A TT
INTENSITY
INTENSITY
INTENSITY
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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)
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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
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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
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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
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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.
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Visit us at http://solcap.msu.edu/