Transcript Marker Assisted Selection in Soybean

Marker Assisted
Selection in
Soybean
Brian Diers
University of Illinois
Mike Lee states in Advances in
Agronomy (1995):
“The challenges loom large for
marker assisted selection (MAS);
in many crops, conventional
selection has had several decades
to evolve into a very effective
technology.”
Outline
• Overview of the soybean industry.
• Marker assisted selection (MAS) in the private
sector.
• Experiences mining for new genes using MAS in
my program.
– Soybean cyst nematode (SCN)
– Aphid resistance
– Yield
• Intellectual property concerns.
• Conclusions.
Take Home Messages
• MAS is being done in the private sector on
a large scale.
• Public breeders are using it successfully in
discovery, prebreeding, and limited cultivar
development.
• Pick your trait well, MAS is being used
successfully for simply inherited traits
(defensive traits).
US Soybean Germplasm has a
Narrow Base
• Breeders in North America are selecting within
an narrow germplasm base.
– 80% can be traced to 13 ancestral lines.
– 50% of the northern US germplasm can be traced to
3 ancestral lines.
• There are likely genes in plant introductions
(PIs) that could be used to improve soybean
yield and other traits.
– The germplasm collection in Urbana contains over
18,000 PIs.
3000
44.6
2500
37.2
2000
29.7
1500
22.3
1000
14.9
500
1920
1930
1940
1950
1960
1970
Production Year
LIN: b = 22.6kg/ha or 0.337bu/ac R2 = 0.934
1980
1990
EXP: b = 0.01464 R2=0.927
From Specht et al. Crop Sci. 39:1560-1570
7.4
2000
Seed Yield (bu/ac)
Seed Yield (kg/ha)
USA Soybean Yield Trend (1924-2000)
Soybean Cultivars Purchased by Farmers
are Mostly From Private Industry
Acres of Certified Seed Produced
of Public Cultivars in Illinois
250000
200000
Acres
• In the Midwest, less
than 5% of the
acreage is planted to
public cultivars.
• Over 90% of the
acreage is planted to
Roundup Ready
cultivars.
• Reduced emphasis of
cultivar development
in the public sector.
150000
100000
50000
0
1975
1980
1985
1990
Year
1995
2000
MAS in soybean is being done on an
industrial scale in private industry
MAS in Private Sector
• Monsanto and Pioneer making 100,000’s
of selections / year.
• Pioneer has used an allele specific
hybridization system.
• Largely being done for disease resistance
genes.
Marker Assisted Selection In
Public Sector
• MAS on the order of thousands of
genotypes a year can be done with
relatively inexpensive technology.
• We use simple sequence repeat (SSR or
microsatellite) markers to do several
thousand selections / year.
– There are a 1,000 mapped SSR markers
publicly available.
Collect Leaf Tissue into 96 Well Plate
Add Extraction Solution
Grind Tissue with Paint
Shaker
Do PCR and Load, Run and Score
Gels
MAS for Resistance to Soybean Cyst
Nematodes (SCN)
• Most important disease of
soybean. Estimate yield
loss is 174 million/bu
annually.
• Resistant cultivars are
available to growers,
perception that they yield
less.
• Phenotype can be highly
heritable but is tedious
and expensive to test.
SCN
on roots
5
MAS Selection for SCN Resistance
can be Effective
PI 88788 source selected with Satt309
Selected
25
Not Selected
20
15
10
5
Female Index - Race 3
121-130
111-120
101-110
91-100
81-90
71-80
61-70
51-60
41-50
31-40
21-30
11-20
0
0-10
Number of Lines
30
SCN Resistance from G. soja
• Can we identify new sources
of SCN resistance genes?
G. soja is a logical species to
look for new resistance
genes.
• In collaboration with Prakash
Arelli (USDA-ARS) we
mapped to major SCN
resistance QTL from PI
468916, a G. soja
introduction.
LOD Plots of SCN QTL
SCN Resistance from G. soja
• The G. soja resistance QTL on LG E and
G were backcrossed into soybean through
MAS.
• BC4 population was tested for SCN
resistance and agronomic performance.
• Selecting for SCN resistance from G. soja
in breeding populations.
Greenhouse Resistance of Lines in a
BC4 Population Segregating for G. soja
Resistance
Female Index
120
100
80
60
40
20
0
Res E, Res G
Res E, Sus G
Sus E, Res G
Sus E, Sus G
Resistant classifications
Yield Test of a BC4 Population
Segregating for G. soja Resistance
5 Environments Across 2 Years
Yield
Linkage
Group
P>F
G. soja
Other
G. max
significance
E
0.006
46.6
45.4
G
0.004
46.8
45.4
Later mat.
(0.75 days)
Greater lod.
(0.15 units)
SCN Germplasm Release
• Release of LDX01-1-65 germplasm line.
– The line has two new SCN resistance genes from wild
soybean crossed into it.
– Line was released and requested by 16 private breeders
from 12 companies.
SCN Female Index
Yield
Mat.
Lod.
Ht.
HG
HG
bu/a
date
score
In.
Type 0
Type 2.5.7
A81-356022
38.8
9-15
1.6
41
95
-
LDX01-1-65
42.6
9-18
2.6
41
14
3
Dwight
49.1
9-11
1.0
39
2
55
Soybean Aphid Resistance
• Soybean aphids became a
new soybean pest in North
America in 2000.
• Losses in Illinois and
Minnesota were estimated to
be in excess of $170 million in
2003.
• Resistance to soybean aphid
was identified in Dowling, a
maturity group VIII cultivars
released in Texas in 1978.
• Dowling is not adapted to the
Midwest.
Aphid Resistance
• Crossed Dowling with Loda (susceptible
adapted cultivar) and tested a F2 population and
F2:3 lines for resistance.
• Made bulks of resistant and susceptible plants
and did a bulk segregant analysis to identify
linked markers.
• Found resistance to be single gene and mapped
the gene.
• Using marker assisted selection to aggressively
develop cultivars with this resistance.
Backcrossing Aphid Resistance
x
Loda (Susc) Dowling (Res)
Grow F1plant
March 2004
Test F2 for resistance, map resistance
Backcrossing Aphid Resistance
X
RS
SS
RS
RS
RR
Select phenotypically resistant F2
plants that also are homozygous
resistant for linked markers.
Loda
Produce BC1F1
(March 2004)
X
Select heterozygous
BC1F1 plants based on
markers.
Loda
Produce BC2F1
(July 2004)
Backcrossing Aphid Resistance
X
Loda
Produce BC3F1
(October 2004)
Select heterozygous BC2F1
plants based on markers.
X
Grow BC2F2 plants,
select resistant ones
Loda
Select heterozygous
BC3F1 plants based Produce BC4F1
(January 2005)
on markers.
Backcrossing Aphid Resistance
X
Select heterozygous BC4F1
plants based on markers.
Grow BC4F2 plants,
select resistant ones
Loda
Produce BC5F1
(March 2004)
Select heterozygous BC5F1
plants based on markers.
(June 2005)
Backcrossing Aphid Resistance
• With modest resources, we have been
able to produce BC5F1 plants.
– March 2004 to June 2005
• In the field in 2005 we have:
– BC1F2-derived and F2-derived lines in yield
tests and resistance evaluations in 4 states.
– BC3F2-derived to F2-derived lines in plant
rows.
– BC5F1, BC4F1s (four backgrounds) and F1s
from several other backgrounds.
Backcrossing Aphid Resistance
• Did not use markers to more
quickly recover the recurrent
parent.
– According to theory with a
selection of 5% of the BCF1s each
generation, we could have
selected BC2F1 with a similar
amount of genome recovered as a
BC3F1.
– Theory meets practicality. Difficult
to product large number of F1s in
soybean.
Mapping of Yield QTL
• Narrow genetic base of soybean makes it
likely that there are new genes in soybean
germplasm that could increase yield.
• How to mine these genes?
• In collaboration with Randy Nelson, my
program is mapping yield increasing QTL
from exotic germplasm.
Mapping of Yield QTL
• IA 2008 x PI 468916 (Glycine soja) BC2
populations.
– Set of 5 backcross 2 populations. Each is
predicted to segregate for 25% of the G. soja
genome.
– Did not use advances backcross methods
because of the difficulty in producing F1s.
• Tested populations for yield across 4
environments in 2 years.
Positive Yield QTL from the
Adapted Parent
• Analysis based on field evaluations across
four environments.
Pop
LG LOD
324
330
334
338
338
K
K
C2
E
M
aEstimated
12
18
7
11
9
R2
aa
0.4
0.38
0.4
0.28
0.29
147
205
124
66
64
Mat.
Ht.
Lod
LOD Score
NS
7
NS
NS
12
13
29
20
NS
NS
8
NS
24
11
NS
effect of substituting one allele of one allele of
PI 468916 with one allele of IA2008.
Positive Yield QTL from G. soja
Pop
LG
324
334
334
334
E
D1b
L
N
aEstimated
Additive Effects a and Probabilities
Yield
Height
Maturity
Lodging
kg ha-1
cm
day
score (1-5)
-53*
ns
ns
ns
-62*
ns
2.4*
ns
-60*
ns
ns
-0.14**
-61*
ns
ns
ns
effect of substituting one allele of one allele of
PI 468916 with one allele of IA2008.
Confirmation Testing
• Confirmation populations were developed prior the the
completion of the study.
• Random BC2 F2 plants from the population were
crossed with another cultivar.
• Populations of F4-derived lines were developed from
these F1s.
• Four populations had regions carrying three ‘moderately
significant’ G. soja regions with a positive effect.
• The G. soja regions were not significant in three
populations but was significant in the fourth.
Confirmation Testing
Linkage Group L QTL
Location
G. soja
G. max
Add. Eff.
Pr>F
Chile
---------kg ha-1---------3687
3077
305
0.05
Urbana, IL
2599
2384
108
0.10
Lincoln, NE
3407
3011
198
0.009
Mead, NE
3546
3381
82
0.45
Mean (no
Chile)
3170
2767
202
0.003
• The confirmation population was small (50 F4-derived
lines)
NIL Development
Determine which of the F4-derived lines from are
heterogeneous for the yield QTL region based on markers.
F4 plant would be fixed for
7/8 of the genome
Linkage Group L QTL in NIL
• Two populations of near isogenic lines
segregating for the LG L QTL were
developed.
• These were tested in 2003 and no
significant marker-yield associations were
found.
Linkage Group E QTL
• LG E QTL was backcrossed into A81356022 because this region is associated
with SCN resistance.
• The additive effect of the G. soja QTL
allele was 1.2 bu/acre yield increase
across two environments in 2004.
• Increase because of SCN resistance?
Need to quantify the SCN pressure in the
fields.
Difficulty
Difficulty
Mining New Genes from Germplasm
Collections
Less
Complex
Inheritance
More
Complex
Inheritance
Trait at a low Trait at a high
level in elite level in elite
Yield QTL vs. Resistance QTL
Selection in Elite Germplasm
SCN
Resistance
Yes
Know how the QTL entered
the gene pool and understand
its movement into elite
germplasm?
Know the allele carried by
Yes
parents based on phenotype?
Yield
Probably
not
No
Intellectual Property
• Non-science issues can cloud the use of
MAS in breeding programs.
• Major company has a patent on the major
SCN resistance gene.
– The royalty rate for using this technology
makes it not economical.
• Public sector moving in the same
direction.
– Pressure from central administration for Ag
Colleges to increase there revenue.
Intellectual Property
• Is the public sector moving to the dark side
with patenting?
I must be true to my
profession and work
for the public good.
Young plant breeder
You will have riches
if you join me and
patent your gene.
Intellectual Property Manager
Conclusions
• Private sector is doing MAS on an industrial
level.
• Public sector does MAS in research and
breeding programs.
• Discovery of useful alleles and MAS for these
alleles has been more successful for simply
inherited traits (resistance) than complex traits
(yield).
• Intellectual property issues may both hinder and
help science.
Acknowledgements
Funding from:
United Soybean Board
Illinois Soybean
Checkoff Board
North Central Soybean
Research Program
USDA-NRI
Contributors:
Shawn Carlson
Randall Nelson
Glen Hartman
Eileen Kabelka
Dechun Wang
Sources of Resistance in
Northern Varieties (MG I-IV)
12 1
29
3
10
PI88788
PI88788 and Peking
Peking
PI209332
PI88788 & PI437654
Peking & PI437654
705
Data from Marion Shier “Soybean varieties with soybean cyst
nematode resistance, January 2002.”