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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 IRRI MAS CASE STUDY

Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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Page 1: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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

IRRI MAS CASE STUDY

Page 2: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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

Page 3: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

‘Mega varieties’

• Many popular and widely-grown 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

Page 4: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Backcrossing strategy• Adopt backcrossing strategy for incorporating

genes/QTLs into ‘mega varieties’• Utilize DNA markers for backcrossing for greater

efficiency – marker assisted backcrossing (MAB)

Page 5: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Conventional backcrossingx P2P1

DonorElite cultivarDesirable trait

e.g. disease resistance

• High yielding

• Susceptible for 1 trait

• Called recurrent parent (RP)

P1 x F1

P1 x BC1

P1 x BC2

P1 x BC3

P1 x BC4

P1 x BC5

P1 x BC6

BC6F2

Visually select BC1 progeny that resemble RP

Discard ~50% BC1

Repeat process until BC6

Recurrent parent genome recovered

Additional backcrosses may be required due to linkage drag

Page 6: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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

Page 7: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Donor/F1 BC1

c

BC3 BC10

TARGET LOCUS

RECURRENT PARENT CHROMOSOME

DONOR CHROMOSOME

TARGET LOCUS

LIN

KE

D D

ON

OR

G

EN

ES

Concept of ‘linkage drag’ • Large amounts of donor chromosome remain even after many backcrosses• Undesirable due to other donor genes that negatively affect agronomic performance

Page 8: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Conventional backcrossing

Marker-assisted backcrossing

F1 BC1

c

BC2

c

BC3 BC10 BC20

F1

c

BC1 BC2

• Markers can be used to greatly minimize the amount of donor chromosome….but how?

TARGET GENE

TARGET GENE

Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection: new tools and strategies. Trends Plant Sci. 3, 236-239.

Page 9: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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

RECOMBINANT SELECTION

1 2 3 4

Page 10: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

OR

Step 1 – select target locus

Step 2 – select recombinant on either side of target locus

BC1

OR

BC2

Step 4 – select for other recombinant on either side of target locus

Step 3 – select target locus again

* *

* Marker locus is fixed for recurrent parent (i.e. homozygous) so does not need to be selected for in BC2

Page 11: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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

Page 12: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Background selection

Percentage of RP genome after backcrossing

Theoretical proportion of the recurrent parent genome is given by the formula:

Where n = number of backcrosses, assuming large population sizes

2n+1 - 1

2n+1

Important concept: although the average percentage of the recurrent parent is 75% for BC1, some individual plants possess more or less RP than others

Page 13: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

P1 x F1

P1 x P2

CONVENTIONAL BACKCROSSING

BC1 VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY RESEMBLE RECURRENT

PARENT

BC2

MARKER-ASSISTED BACKCROSSING

P1 x F1

P1 x P2

BC1 USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE MOST RP MARKERS AND SMALLEST %

OF DONOR GENOME

BC2

Page 14: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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

Page 15: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Screening for submergence tolerance

Page 16: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

A major QTL on chrom. 9 for submergence tolerance – Sub1 QTL

1 2 3 4 5 6 7 8 90

5

10

15

20

Submergence tolerance score

IR40931-26 PI543851

Segregation in an F3 population

0 10 20 30 40

LOD score

50cM

100cM

150cM

OPN4

OPAB16

C1232

RZ698

OPS14RG553

R1016RZ206

RZ422

C985

RG570

RG451

RZ404

Sub-1(t)

1200

850

900

OPH7950

OPQ1600

Xu and Mackill (1996) Mol Breed 2: 219

Page 17: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Make the backcrosses

SwarnaPopular variety

X

IR49830Sub1 donor

F1 X Swarna

BC1F1

Page 18: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Pre-germinate the F1 seeds and seedthem in the seedboxes

Seeding BC1F1s

Page 19: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Collect the leaf samples - 10 days after transplanting for marker analysis

Page 20: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Genotyping to select the BC1F1 plants with a desired character for crosses

Page 21: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Seed increase of tolerant BC2F2 plant

Page 22: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Selection for Swarna+Sub1

Swarna/IR49830 F1

Swarna

BC1F1697 plants

Plant #242

Swarna

376 had Sub121 recombinantSelect plant with fewest donor alleles

158 had Sub15 recombinant

SwarnaPlant #227

BC3F118 plants

1 plant Sub1 with2 donor segments

BC2F1320 plants

Plants #246 and #81

Plant 237BC2F2

BC2F2937 plants

Page 23: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Time frame for “enhancing” mega-varieties

May need to continue until BC3F2

• Name of process: “variety enhancement” (by D. Mackill)

• Process also called “line conversion” (Ribaut et al. 2002)

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.

Page 24: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Swarna with Sub1

Page 25: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

Graphical genotype of Swarna-Sub1

BC3F2 lineApproximately 2.9 MB of donor DNA

Page 26: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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

Page 27: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

IBf locus on tip of chrom 9:inhibitor of brown furrows

Page 28: Marker-assisted backcrossing for submergence tolerance David Mackill, Reycel Mighirang-Rodrigez, Varoy Pamplona, CN Neeraja, Sigrid Heuer, Iftekhar Khandakar,

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