Plant Breeding in the Genomics Era

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Plant Breeding in the Genomics Era

• D.S. Brar
• IRRI

29 May 2008
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• Plant Breeding is the art and science of
  improving the heredity (genetic properties) of
  plants to produce superior varieties and products
  in a sustainable and environment- friendly way.
• Plant Breeding comprises of two phases
• Evolutionary phase where variable populations
  are created (e.g IRRI has made 88,631 crosses).
• Evaluationary phase superior genotypes are
  selection based on evaluation
• Why Plant Breeding is still an art?

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Objectives

1. Higher yield potential
2. Multiple resistance to pests (diseases, insects)
3. Tolerance to major abiotic stresses (drought,
   submergence, salinity, cold etc.)
4. Superior grain and nutritional quality
   (micronutrient enriched)- high iron, high zinc,
   pro-vit A etc.
5. Varieties less vulnerable to climatic changes

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Rice production- challenges

• World rice production has more than doubled (256
  million in tonnes) in 1965 to 600 million tonnes
  (2006)
• To meet the growing need of increasing human
  population 25 more rice needed by 2020
• How to increase production with less land, less
  water, less chemicals and less labor ?
• How to sustain rice production under the emerging
  global climatic changes ?

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Breeding approaches

• 1. Conventional breeding
• 2. Ideotype breeding (Physiological breeding)
• 3. Heterosis (hybrid) breeding
• 4. Wide hybridization
• 5. Induced mutation
• 6. Dihaploid and somaclonal variation

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Resources for genomics research

• Dense molecular map (4,000 DNA markers)
• Novel genetic resources for functional genomics
• 1. T-DNA insertion lines
• 2. deletion mutants
• 3. YAC, BAC, EST libraries
• High throughput methods/ materials (gene chips,
  microarray) for gene discovery and gene
  expression analysis
• High throughput transformation protocols/materials
  - validation of candidate genes and producing
  transgenics efficiently
• Comparative genetic maps (synteny across
  genomes/species)

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Specialized genetic stocks for future research in
genomics and Plant Breeding

1. Near Isogenic lines (NILs)-BB, blast, BPH(?),
   tungro(?), abiotic stresses (submergence,??)
2. Recombinant inbred lines (RILs)
3. Backcross- RILs
4. Dihaploids
5. Chromosome segmental substitution lines
   (CSSL)-indica, japonica, wild species

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Mendelian segregation for the disease resistance
and molecular marker
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Molecular markers and genomics- perspectives in
plant breeding

• Mapping of genes and QTLs governing agronomic
  (complex) traits- more input for difficult traits
  (sheath blight, stemborer etc.)
• MAS (marker/gene based) -gene pyramiding for
  enhancing tolerance to various stresses
• Transgenic breeding (GM rice)-new genes/
  transcription factors needed
• (search for orthologs in rice germplasm)
• 4. Gene discovery- candidate genes- mining
  novel alleles
• 5. Functional genomics-reverse genetics-
  understanding gene function and transferring such
  genes into elite breeding lines (grain quality of
  IR64?)

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Major success stories in Plant Breeding
Trait Impact
Dwarfing genes sd1 Semi-dwarf varieties -green revolution (256m tons to 600m tons)
Male sterility O. sativa f. spontanea lt90 hybrids rice worldwide has this CMS source
Early maturity (short duration) Increased cropping intensity- 2 to 3 crops a year.
Pest resistance Many varieties with multiple resistance to pests developed reduce use of chemicals and increase yield stability (O. nivara only source of grassy stunt resistance
Tolerance to abiotic stresses Many varieties resistant to abiotic stresses developed-yield stability
50 dwarfing genes 7 sd genes
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Use of dwarfing genes in rice breeding
Success story 1
Cross Variety Institute
Dee-Geo-Woo-Gen x Tsai-yuan-chung (1949) TN-1 (1956) Taichung District Agricultural Improvement Station, Taiwan
Ai-zai-zhan x Guang-chung 13 Guang-chang-ai (1959) Mainland China
Dee-Geo-Woo-Gen x Peta IR8 (1966) IRRI
sd1 gene used worldwide in breeding dwarf
varieties
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Developing short duration varieties
Success story 2
Traditional varieties Donor Todays cultivars Impact
150-180 days TKM6 (India) 95-120 days Cropping intensity increased -possible to grow 2 or 3 rice crops in a year
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Success story 3
Hybrid rice breeding

• A male sterile wild rice plant with abortive
  pollen found in 1970
• and designated as wild abortive (WA)
• used as parent- lt90 hybrid rice grown worldwide
• 15.8 million hectares in China
• 2.5 million hectares in countries other than
  China

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Some genes for pest resistance identified through
classical genetic analysis
Pest Genes identified
BB (30 genes) Blast Xa1,Xa2,Xa3,Xa4,xa5,Xa7,xa8,Xa10, Xa11,Xa12, xa13,Xa14,xa15,Xa16, Xa17, Xa18,xa19,xa20,Xa21,Xa22, xa24,Xa25,Xa26,Xa27,xa28,Xa29 (40 genes)
BPH Bph1,bph2,Bph3,bph4, bph5,bph6, bph7,Bph9,bph8, Bph10, bph11, bph12, bph13, Bph14, bph15, Bph16, Bph17, Bph18t, bph19
GLH Glh1, Glh2, Glh3, glh4, , Glh5, Glh6, Glh7, Glh8, Glh9, glh10
Gall midge Gm1, Gm2, gm3, Gm4, Gm5, Gm6, Gm7, Gm 8, Gm9, Gm10

• These genes are the backbone for the application
  of molecular markers and genomics and for
  breeding pest resistant varieties

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IR varieties with multiple resistance to diseases
and insects
Success story 4
Variety Blast BB Grassy stunt Tungro GLH BPH biotypes BPH biotypes BPH biotypes Stem borer Gall midge
Variety Blast BB Grassy stunt Tungro GLH 1 2 3 Stem borer Gall midge
IR8 MR S S S R S S S MS S
IR24 S S S S R S S S S S
IR26 MR R MR MR R R S R MR S
IR36 R R R R R R R S MR R
IR64 MR R R R R R R MR R MR
IR72 MR R R R R R R R MR -
IR74 R S R R R R R R MR -
Many varieties with resistance to biotic stresses
have been released by NARES worldwide
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Success story 5

• IR varieties with tolerance to adverse soils

Variety Wetland soils Wetland soils Wetland soils Wetland soils Wetland soils Wetland soils Wetland soils Dryland soils Dryland soils
Variety Toxicities Toxicities Toxicities Toxicities Toxicities Deficiencies Deficiencies Deficiency Toxicities
Variety Salt Alkali Peat Iron Boron Phosphorus Zinc Iron Aluminum and manganese
IR8 4 6 5 8 4 4 4 4 4
IR36 3 3 3 3 3 6 3 2 2
IR64 3 3 4 5 4 4 4 0 0
IR72 6 5 0 3 0 5 4 0 0
IR74 4 5 0 3 0 5 5 0 0
0 no information, 1 almost normal plants 9
plants almost dead
Several varieties tolerant to abiotic stresses
have been released worldwide- yield stability
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• 328 IRRI breeding lines have been released as
  643 varieties in 75 countries

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Enhancing yield potential
Inbred and hybrid rice breeding
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Three NPT lines (japonica type) released in the
Yunnan province of China, IR64446-7-10-5
Dianchao 1 (2002) IR69097-AC2-1 Dianchao 2
(2003) IR64446-7-10-5 Dianchao 3 (2000)
Promising elite indica type with 10-15 higher
yield than IR72
IR77186-122-2-2-3 1st NPT line from a cross of
tropical japonica x indica released as variety
(NSICRc 158) in the Philippines in 2007
Virk unpublished
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Progress in developing high-yielding hybrid rice
varieties

• 15.8 million hectares in China
• 2.5 million hectares in countries other than
  China
• Gain in yield 10- 15 over best inbred
  cultivar(s)

Courtesy F. Xie
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Wide hybridization
Genes for resistance to BPH (Bph10, Bph18,
Bph(t), BB (Xa21 unknown), blast (Pi9, Pi40),
tungro, grassy stunt and tolerance to acid
sulfate, iron toxicity(?) and CMS have been
transferred from wild species into rice
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IRRI breeding lines released as varieties
through wide hybridization
Tungro disease (Philippines) O. rufipogon
BPH resistant (Vietnam) O. officinalis
NSICRc112
High yielding (Philippines) O. longistaminata
A popular variety in rice grown on 100,000 ha in
Mekong Delta ( Vietnam) O. rufipogon
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Yield enhancing loci (yld 1 and yld 2) identified
in wild species (9 publications)
IRRI, CIAT, many institutes in Japan, China are
developing CSSL for mapping genes/QTLs and for
use in functional genomics/breeding
shaded donor chromosome segments
Chromosome segmental substitution lines - CSSL
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MAAL
Autosyndetic (AA, EE) and allosyndetic (AE)
pairing as detected through (GISH)
IRRI (unpublished)
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Molecular markers and their application in plant
breeding
A dense molecular map (4000 DNA markers)
Conventional genetic map (lt250 markers)
SNP markers in rice Monna et al. 2006, DNA
Research

• Many genes/ QTL tagged map based gene cloning
  possible
• MAS became possible to accelerate breeding
• Gene pyramiding to enhance tolerance to stresses

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Mapping of centromeres on rice chromosomes using
cytogenetic stocks and RFLP markers
1st report on centromere mapping of molecular
map- reference to identify genes on short or long
arms of rice chromosomes
PNAS (1996) 936163-6168
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Some examples of rice genes of agronomic
importance tagged with molecular markers
Trait Gene(s)
BB resistance Xa1, Xa2, Xa4, xa5, Xa10, xa13, Xa21, Xa22
Blast resistance Pi1, Pi2, Pi4, Pita, Pi6, Pi7, Pi9, Pi10, Pi11, Pi12, Pib, Pi2, Pikm, Pb1
BPH resistance Bph1, bph2, Bph9, Bph10, Bph18, Qbph1, Qbph2
Gall midge resistance Gm1, Gm2, gm3, Gm4, Gm5, Gm6
Fertility restoration Rf1, Rf2, Rf3, Rf5
Thermosensitive male sterility tm1, tm3, tms4
Photoperiod sensitive male sterility pms1, pms2, pms3
P deficiency Pup1
Drought QTLs mapped
Submergence Sub 1
Salt tolerance SolT
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Molecular marker assisted selection and gene
pyramiding practiced in rice
Trait Gene(s) Countries
Bacterial blighta Xa4, xa5, xa7, xa13, Xa21 Philippines, China, India, Indonesia, Korea
Blast Pi, Piz, P1b, Pikh India, China, Japan, Korea
Gall midge Gm1, Gm2, Gm4, Gm6 China, India
BPH Bph18 Korea
Submergence Sub1 IRRI, India, Bangladesh and now being extended to South-East Asia
Salt SolT ?
Drought QTLs ?
P-deficiency Pup1 ?
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MAS products released as rice varieties for
cultivation in Philippines, India, Indonesia and
China
Variety Genes for BB resistance Country Country
NSIC142  ( PR31563-AR32-19-3-3) Xa21 others Xa21 others Philippines
NSIC154 ( PR31561-AR32-11-83-1-4-1) Xa21others Xa21others Philippines
Improved Pusa Basmati Improved Samba Mahsuri Xa21 others Xa21 others Xa21 others Xa21 others India India
Three commercial hybrids Xa21 others Xa21 others China
Angke Xa4 xa5 Xa4 xa5 Indonesia
Conde Xa4 Xa7 Xa4 Xa7 Indonesia
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Developing a submergencetolerant version of
Swarna, a widely grown variety
Swarna intolerant
IR49830-7 tolerant
Marker-assisted backcrossing

• Target gene selection
• Background selection

Sub1
BC2 or BC3
Swarna-Sub1
Courtesy D.J. Mackill ( see details on Sub 1,
Nature,2006, Vol 442, 705-708)
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Identification of major QTLs for yield under
drought- RM 511 on Chr. 12
10cM (3.3 Mb)
RM28048 RM511

• Future research
• Transferring such QTLs into high yielding
  varieties through MAS
• Pyramiding of QTLs with different mechanisms of
  drought tolerance

Bernier et al (2007), Crop Sci. 47 507-518
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MAS used to transfer aroma in elite breeding lines
Bradbury et al (2005) Molecular Breeding 16
279283
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Some examples on GM rice carrying agronomically
important genes
Rice Transformation
Trait Transgene Transformation method(s)
Herbicide resistance bar PEG, Biolistic
Tolerance to virus coat protein Electroporation, Biolistic
Sheath blight resistance Chitinase PEG
Stemborer resistance cry1(A), cry1(B), cry1(C ) Electroporation, Biolistic, Agrobacterium
CpTi PEG
Tolerance to BPH gna Biolistic
BB resistance Xa21 Biolistic
Blast resistance Afp Agrobacterium
Salt tolerance cod A Electroporation
Drought tolerance OtsA, OtsB, DREB1A Agrobacterium
Increased iron content ferritin Agrobacterium
Pro-vit A golden rice psy, crtl, lcy psy (maize), crtl Agrobacterium Agrobacterium
Reduced O2 inhibition of photosynthesis PEPC (C4 enzyme from maize) Agrobacterium
Field tested in India and China
modified from Brar and Khush (2002)
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Wild type
Np Psylcrtl
Zm Psylcrtl
Wild type
Transgenic golden rice with genes from maize,
daffodil and bacteria (Erwinia sp.)
Source Nature Biotechnology (2005) 23482-487
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IR64 introgression lines with beta carotene locus
(BC2F4 Field testing in DS2008)
MAS in transgenic breeding
Polished rice grains of variety Cocodrie and
transgenic Cocodrie event 309
Courtesy of P.S. Virk and G. Barry
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Expectations of Plant Breeders from genomics
research

• Identification of genes which can enhance yield
  potential (Ghd7- nature genetics 2008, glgC16-
  modifying starch biosynthesis)
• Identification of heterotic gene blocks to
  enhance level of heterosis
• Introgression of yield enhancing loci/QTLs, wild
  species alleles into high yielding
  cultivars/hybrids
• MAS (marker/ gene based) and pyramiding of
  genes/QTLs with different mechanisms to enhance
  tolerance to biotic and abiotic stresses
• Pyramiding of transgenes, Bt gene
• Drought tolerance, transcription factors (DREB,
  HRD (HARDY)

continued
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Expectations of Plant Breeders from genomics
research

• Gene discovery to identify novel alleles for
  agronomic traits e.g submergence, disease
  resistance etc.
• Developing apomictic rice- identification and
  transfer of genes from the existing apomictic
  species (300 plant species are apomictic in
  nature)
• Identification of pairing controlling genes (like
  Ph gene in wheat) to promote introgression of
  useful genes from distant genomes of wild species
  into rice26

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Conclusions

• Genomic research is an important supplement to
  conventional breeding ( both in evolutionary and
  evaluationary phases of breeding) but not a
  substitute.
• Integration of molecular marker technology and
  genomics with conventional plant breeding is
  emphasized to develop high yielding rice
  varieties tolerant to major biotic and abiotic
  stresses with improved grain quality and
  nutritive value
• Appropriate phenotying and experimental
  designs/trials are emphasized for QTL mapping and
  use in MAS
• Continued efforts are needed to develop high
  throughput and cost effective genomic tools for
  use in plant breeding.
• Need for new generation of plant breeders

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Thanking you for the kind attention