PCR-Based Markers for Oil, b-glucan, and Protein Content

1
PCR-Based Markers for Oil, b-glucan, and
Protein Content
Steve Molnar, Winson Orr, Julie Chapados, Anissa
Lybaert, Ismahane Elouafi, Shan He, Charlene
Wight, Diane Mather, and Nick Tinker Eastern
Cereal and Oilseed Research Centre, Ottawa and
McGill University
Presented to American Oat Workers
Conference Fargo, North Dakota July 23-26, 2006
2
Why Are We Interested?

• Oil, b-glucan, and protein content are three
  major quality traits in oats.
• One strategy for improving these traits is to use
  molecular marker assisted selection (MAS)
  breeding.
• For MAS, PCR-based markers are currently the best
  choice balancing low cost with high reliability.

3
The Challenge

• Identify the genetic loci that are responsible
  for variation in these 3 traits.
• Identify or develop PCR-based markers linked to
  these loci.
• Preferably, identify or develop PCR-based markers
  within the gene itself, as these can not be
  unlinked by recombination and as these could be
  allele specific.

4
Oil QTLs Reported in Mapping Populations
KO LG KO QTL KM QTL TM QTL OM QTL Iowa Linked QTLs Gene ?

KO11 KO11 KM11 OM3 bg, protein Accase A
KO37 KO37
KO ? Bcd1829y protein
Unlinked Coleoptal
KO6 (KO6) bg
KO22 KM22 TM15 protein
KO ? KM5X bg
KO24_26_34 TM5 bg, protein N1 naked
KO32 TM4_16
KO ? TM30
KO2 OM6
KO ? OM13
KO ? OM19
KO ? OM20
KO ? OM24
KO 30 OT16
Explained 45-48 22-28 39 42-52
5
The Challenge

• Identify the genetic loci that are responsible
  for variation in these traits
• Identify or develop PCR-based markers linked to
  these loci
• Preferably, identify or develop PCR-based markers
  within the gene itself, as these can not be
  unlinked by recombination and as these could be
  allele specific

6
PCR-Based Markers Proven as Linked to Oil, bG, or
Protein QTLs in Map. Pops. or as Candidate Genes
Trait Pop RFLP AFLP RAPD RAPD SSR Gene EST Total
QTLs SCAR SCAR ISSR Primers Primers

Oil KO 3-4 Many 1 1
KM 3 Many
TM 5 Many 2 6 2
OM 3-6 Many 1
FR 2 1
DE 2 1
Other ? 2 gt 2
gt 19
b-g KO Many 2 5 1
KM Many
TM Many 2 3 5
18
Pro TM Many 1 6 1
OM Many 1
9
7
PCR Based Markers Linked to Oil QTL
T T M M
R R R R R R R R R R T T M M

RAPD
SCAR
Ethidium bromide-stained gel (2 agarose) of PCR
amplification products after electrophoresis.
RAPD UBC121 and derived SCAR on Terra, Marion and
their RILs.
Orr and Molnar, submitted
8
QTL Linked RAPD Markers and Derived SCARs
A
B
TM15(KO22)
TM11
TM5(KO24,34)
0.0
acc_cta222
Oil
58.3
cdo482x
ß-glucan
0.0
bcd327x
13.4
umn575
aco139y
4.9


bcd327
14.0

62.3
ubc185s
14.3
ubc167a
14.4
ubc198
62.6
ubc185

ubc121
18.9

ubc186, ubc167ts
64.6
n1
Oil, Protein, etc.
15.0
ubc186s
15.2
15.3
ubc121ms
15.5
ubc167ms
ubc198s
16.1
aco139x
C

D
KO15
TM30
KO11_4120
umn5112_2

19.3

cdo460b
6.2
0.0
acc_cat259
7.0
ACCase 1
Oil
isu1507xp
12.8
7.1
cdo665b

ubc364os
33.9
umn5214

7.8


43.0
cdo54
bcd115x
Oil
32.0


10.1
48.4
ubc364b

cdo1436c
Orr and Molnar, submitted
51.6
bcd115c

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SCAR UBC364Os Tested On Dal (High Oil) x Exeter
(Low Oil) Recombinant Inbred Lines
2-11 High Oil RILs 12-19 Low Oil
Kanota
Ogle
LDD EE KK OO
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PCR-Based Markers Proven as Linked to Oil, bG, or
Protein QTLs in Map. Pops. or as Candidate Genes
Trait Pop RFLP AFLP RAPD RAPD SSR Gene EST Total
QTLs SCAR SCAR ISSR Primers Primers

Oil KO 3-4 Many 1 1
KM 3 Many
TM 5 Many 2 6 2
OM 3-6 Many 1
FR 2 1
DE 2 1
Other ? 2 gt 2
gt 19
b-g KO Many 2 5 1
KM Many
TM Many 2 3 5
18
Pro TM Many 1 6 1
OM Many 1
9
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Microsatellite (SSR) Markers

• Microsatellite or SSR (Simple Sequence Repeat)
  markers target variation in small repeat units,
  typically 2 or 3 bp in size.
• Graham Scoles lab and Les Domiers lab isolated
  oat SSRs. Polymorphism is extremely low (5).
  However, they and we have tested over 100 and
  mapped all polymorphic ones. Of those, most are
  /-SCAR type markers, not typical co-dominant
  SSRs. This reflects variation in primer binding
  site, not in the number of repeat units. Two
  link to oil QTLs and 6 to b-glucan.

12
Microsatellite (SSR) Markers

• Heidi Kaepplers lab has developed 2 SSRs linked
  to oil and protein QTLs respectively
• Graham Scoles lab has identified 2 ISSR (Inter
  SSR) markers, linked to b-glucan QTLs.
• Studying the Iowa high oil selection program,
  Jean-Luc Jannink has identified 2 SSRs (1 from
  Jim Holland and 1 from Graham Scoles) associated
  with high oil.
• Recently, Nick Tinkers lab has developed new
  SSRs of which 2 are linked to oil, 2 to b-glucan
  and 1 to protein.

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Challenge

• Identify the genetic loci that are responsible
  for variation in these traits
• Identify or develop PCR-based markers linked to
  these loci
• Preferably, identify or develop PCR-based markers
  within the gene itself, as these can not be
  unlinked by recombination and as these could be
  allele specific

14
PCR Cloning of Candidate Genes

• In this approach, we proposed candidate genes
  based on metabolic pathways. Available grass DNA
  sequences for each gene were compared and PCR
  primers designed. These were used to isolate the
  oat homolog, which can then be mapped and its map
  location compared to that of known QTL.

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Candidate Gene Primers

• Anissa Lybaert in our lab found that most PCR
  primers developed this way are monomorphic.
• Catherine Howarth et al have had success with
  this strategy, in part by going one step further
  to identify sequence differences in those PCR
  products and to design SNP (Single Nucleotide
  Polymorphism) markers targeting them. They have
  developed new PCR-based markers for ACCase and
  KAS and other oil biosynthesis enzymes.

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EST Primers

• EST (Expressed Sequence Tag) libraries developed
  from tissues actively involved in oil, b-glucan
  or protein synthesis might be a rich source of
  genes.
• PCR-primers for such ESTs is a potential
  strategy.
• Ismahane Elouafi in my lab recently developed 73
  primer pairs to oat ESTs. We targeted those oat
  ESTs with homology to rice genomic sequences with
  introns, in an effort to increase polymorphism.
  However, only 1 was polymorphic on KxO, even
  after restriction digestion.
• By chance, that EST mapped near a b-glucan QTL.
• However, low polymorphism may limit this approach.

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PCR-Based Markers Proven as Linked to Oil, bG, or
Protein QTLs in Map. Pops. or as Candidate Genes
Trait Pop RFLP AFLP RAPD RAPD SSR Gene EST Total
QTLs SCAR SCAR ISSR Primers Primers

Oil KO 3-4 Many 1 1
KM 3 Many
TM 5 Many 2 6 2
OM 3-6 Many 1
FR 2 1
DE 2 1
Other ? 2 gt 2
gt 19
b-g KO Many 2 5 1
KM Many
TM Many 2 3 5
18
Pro TM Many 1 6 1
OM Many 1
9
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Conclusions

• This is a very conservative inventory of markers
  since it is limited to PCR-based markers mapped
  in the same population as the QTL or derived from
  candidate genes or ESTs. Excellent markers in
  homologous regions of other mapping populations
  or in homeologous regions of all populations have
  not been added to this inventory.
• We need more common markers to better define
  homologous and homeologous relationships and
  ideally a consensus map
• A good selection of PCR-based markers have been
  developed for oil, b-glucan, and protein content
  by numerous laboratories.
• We need databases to manage these and make them
  readily available to the community.

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Conclusions

• These markers are of many different types, that
  have different features. This increases
  flexibility but is problematic for automation.
• Further, the low frequencies of polymorphism in
  oats mean that multiple markers must be developed
  per locus to ensure that an informative marker is
  available in the breeders germplasm of choice.
• We need a single type of marker that is robust,
  numerous and polymorphic.

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Conclusions

• It is unclear if sufficient markers exist because
  it is unclear how many loci are important and
  need to be tagged with markers.
• To determine the number of unique loci we need to
  better define homologous and homeologous
  relationships.
• Regarding the identification of important loci,
  it is noteworthy that QTLs identified for
  b-glucan explain only 20-25 of the parental
  variation. Values are better for protein
  (20-50) and best for oil (35-70).

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Major Conclusions

• It is clear that sufficient high quality markers
  exist to initiate MAS for these three traits,
  when appropriate.
• It is also clear that the mapping of QTLs and
  candidate genes has increased our knowledge of
  the biology and genetics of these traits, which
  in turn affects the design of all oat improvement
  strategies, whether they use markers for MAS or
  not.

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Acknowledgements

• We thank
• Quaker Oats (USA), a division of Pepsico, and QTG
  (Quaker/Tropicana/Gatorade) Canada for many years
  of generous financial support.
• In particular, Fran Webster, Sam Weaver, Bruce
  Roskens and Grant Morrison, our coordinators from
  Quaker.
• Members of the oat team at ECORC, AAFC, Ottawa
  for their dedication to oat improvement.
• as well as Agriculture and Agri-Food Canadas
  Matching Investment Initiative.

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Oat Team and Lab-Mates