Transcript Markers, QTL mapping and marker

IRRI MAS CASE STUDY
Marker-assisted backcrossing for
submergence tolerance
David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona,
CN Neeraja, Sigrid Heuer, Iftekhar Khandakar, Darlene
Sanchez, Endang Septiningsih & Abdel Ismail
Abiotic stresses are major constraints
to rice production in SE Asia
• Rice is often grown in unfavourable
environments in Asia
• Major abiotic constraints include:
–
–
–
–
Drought
Submergence
Salinity
Phosphorus deficiency
• High priority at IRRI
• Sources of tolerance for all traits in germplasm and
major QTLs and tightly-linked DNA markers have been
identified for several traits
‘Mega varieties’
• Many popular and widelygrown rice varieties - “Mega
varieties”
– Extremely popular with farmers
• Traditional varieties with
levels of abiotic stress
tolerance exist however,
farmers are reluctant to use
other varieties
– poor agronomic and quality
characteristics
BR11
Bangladesh
CR1009
India
IR64
All Asia
KDML105
Thailand
Mahsuri
India
MTU1010
India
RD6
Thailand
Samba
Mahsuri
India
Swarna
India,
Bangladesh
1-10 Million hectares
Backcrossing strategy
• Adopt backcrossing strategy for incorporating
genes/QTLs into ‘mega varieties’
• Utilize DNA markers for backcrossing for greater
efficiency – marker assisted backcrossing (MAB)
Conventional backcrossing
• High yielding
• Susceptible for 1
trait
P1
Elite cultivar
x
P2
Donor
Desirable trait
e.g. disease resistance
P1 x F1
• Called recurrent
parent (RP)
P1 x BC1
P1 x BC2
Discard ~50% BC1
Visually select BC1 progeny that resemble RP
Repeat process until BC6
P1 x BC3
P1 x BC4
P1 x BC5
P1 x BC6
BC6F2
Recurrent parent genome recovered
Additional backcrosses may be required due to linkage drag
MAB: 1ST LEVEL OF SELECTION –
FOREGROUND SELECTION
• Selection for target gene or
QTL
• Useful for traits that are difficult
to evaluate
• Also useful for recessive genes
1
2
3
4
Target locus
TARGET LOCUS
SELECTION
FOREGROUND SELECTION
Concept of ‘linkage drag’
TARGET
LOCUS
c
Donor/F1
BC1
BC3
RECURRENT PARENT
CHROMOSOME
DONOR
CHROMOSOME
TARGET
LOCUS
BC10
LINKED DONOR
GENES
• Large amounts of donor chromosome remain even after
many backcrosses
• Undesirable due to other donor genes that negatively
affect agronomic performance
• Markers can be used to greatly minimize the amount
of donor chromosome….but how?
Conventional backcrossing
TARGET
GENE
F1
BC1
c
c
BC2
BC3
BC10
BC20
Marker-assisted backcrossing
TARGET
GENE
c
Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection:
new tools and strategies. Trends Plant Sci. 3, 236-239.
F1
BC1
BC2
MAB: 2ND LEVEL OF SELECTION RECOMBINANT SELECTION
• Use flanking markers to
select recombinants
between the target locus and
flanking marker
• Linkage drag is minimized
• Require large population
sizes
– depends on distance of
flanking markers from target
locus)
• Important when donor is a
traditional variety
1
2
3
4
RECOMBINANT
SELECTION
Step 1 – select target locus
BC1
Step 2 – select recombinant on either side of target locus
OR
Step 3 – select target locus again
BC2
Step 4 – select for other recombinant on either side of target locus
*
*
OR
* Marker locus is fixed for recurrent parent (i.e. homozygous) so does not need to be selected for in BC2
MAB: 3RD LEVEL OF SELECTION BACKGROUND SELECTION
• Use unlinked markers to
select against donor
• Accelerates the recovery of
the recurrent parent genome
• Savings of 2, 3 or even 4
backcross generations may
be possible
1
2
3
4
BACKGROUND
SELECTION
Background selection
Theoretical proportion of
the recurrent parent
genome is given by the
formula:
2n+1 - 1
2n+1
Where n = number of backcrosses,
assuming large population sizes
Percentage of RP genome after backcrossing
Important concept: although the average percentage of
the recurrent parent is 75% for BC1, some individual
plants possess more or less RP than others
CONVENTIONAL BACKCROSSING
P1 x
P2
MARKER-ASSISTED BACKCROSSING
P1 x
P1 x F1
P1 x F1
BC1
BC1
VISUAL SELECTION OF BC1 PLANTS THAT
MOST CLOSELY RESEMBLE RECURRENT
PARENT
BC2
P2
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS
THAT HAVE MOST RP MARKERS AND SMALLEST %
OF DONOR GENOME
BC2
Breeding for submergence tolerance
• Large areas of rainfed lowland
rice have short-term
submergence (eastern India to
SE Asia); > 10 m ha
• Even favorable areas have
short-term flooding problems
in some years
• Distinguished from other types
of flooding tolerance
– elongation ability
– anaerobic germination tolerance
Screening for submergence tolerance
A major QTL on chrom. 9 for
submergence tolerance – Sub1 QTL
0
IR40931-26
PI543851
OPQ1
600
OPN41200
OPAB16
850
20
Sub-1(t)
C1232
RZ698
15
OPS14 900
RG553
R1016
RZ206
50cM
OPH7
950
10
RZ422
5
100cM
C985
0
1
2
3
4
5
6
7
Submergence tolerance score
8
9
RG570
150cM
Segregation in an F3 population
Xu and Mackill (1996) Mol Breed 2: 219
RG451
RZ404
10
LOD score
20
30
40
Make the backcrosses
X
Swarna
Popular variety
IR49830
Sub1 donor
F1 X
Swarna
BC1F1
Seeding BC1F1s
Pre-germinate the F1 seeds and seed
them in the seedboxes
Collect the leaf samples - 10 days after
transplanting for marker analysis
Genotyping to select the BC1F1 plants
with a desired character for crosses
Seed increase of tolerant
BC2F2 plant
Selection for Swarna+Sub1
Swarna/
IR49830 F1
376 had Sub1
21 recombinant
Select plant
with fewest
donor alleles
BC2F2
937 plants
Swarna
Plant #242
BC1F1
697 plants
Plants
#246 and
#81
Swarna
BC2F1
320 plants
Plant #227
158 had Sub1
5 recombinant
Swarna
Plant 237
BC2F2
1 plant Sub1 with
2 donor segments
BC3F1
18 plants
Time frame for “enhancing” megavarieties
• Name of
process: “variety
enhancement”
(by D. Mackill)
• Process also
called “line
conversion”
(Ribaut et al.
2002)
May need to continue until BC3F2
Mackill et al 2006. QTLs in rice breeding: examples for abiotic stresses. Paper presented
at the Fifth International Rice Genetics Symposium.
Ribaut et al. 2002. Ribaut, J.-M., C. Jiang & D. Hoisington, 2002. Simulation experiments on
efficiencies of gene introgression by backcrossing. Crop Sci 42: 557–565.
Swarna with Sub1
Graphical genotype of Swarna-Sub1
BC3F2 line
Approximately 2.9 MB of donor DNA
Swarna
246-237
Percent chalky grains
Chalk(0-10%)=84.9
Chalk(10-25%)=9.1
Chalk(25-50%)=3.5
Chalk(>75%)=2.1
Chalk(0-10%)=93.3
Chalk(10-25%)=2.3
Chalk(25-50%)=3.7
Chalk(>75%)=0.8
Average length=0.2mm
Average length=0.2mm
Average width=2.3mm
Average width=2.2mm
Amylose content (%)=25
Gel temperature=HI/I
Gel consistency=98
Amylose content (%)=25
Gel temperature=I
Gel consistency=92
IBf locus on tip of chrom 9:
inhibitor of brown furrows
Some considerations for MAB
• IRRI’s goal: several “enhanced Mega varieties”
• Main considerations:
– Cost
– Labour
– Resources
– Efficiency
– Timeframe
• Strategies for optimization of MAB process important
– Number of BC generations
– Reducing marker data points (MDP)
– Strategies for 2 or more genes/QTLs