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Mapping of Quantitative Trait Loci (QTL) forGrain Cooking and Eating Quality using aDoubled Haploid (DH) Population from
Anther Culture of Indica/Japonica
Hybrids of Rice (Oryza sativa L.)
VICTORIA CHAVEZ LAPITANToshinori Abe, Edilberto D. Redoña, Darshan S. Brar
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RICE IS LIFE!
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Rice (Oryza sativa L.) is . . .
Most important cereal crop in the worldStaple food for >half of human population
Provides > one fifth of the calories consumedworldwide
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Consumed in the form of noodles,puffed rice, fermented sweet rice,and snack foods
Used in making beer, rice wine,and vinegar
Grain quality: selection criterion in mostrice breeding programs
Key cooking and eating quality traits:
• Amylose content (AC)
• Gel consistency (GC)
• Gelatinization temperature (GT)
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Quality improvement thruconventional breeding is difficult
due to quantitative inheritance
DNA marker technology facilitatesunderstanding of complex quantitativetraits.
Doubled haploid (DH) lines excellent
materials for genetic studies because oftheir homozygosity and uniformity
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OBJECTIVES
To determine the genetic diversity among a set of rice
cultivars
To generate DH lines as mapping population from adiverse cross through anther culture;
To demonstrate the novel use of the DH population forbreeding and genomics through molecular analysis
To identify QTLs for AC, GC, and GT utilizing the DHpopulation
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Rice cultivars
(different quality traits)
Development of crosses
Anther culture of F1s
Doubled haploid (DH) lines
QTL mapping
•Genetic diversity analysis
• Selection of best cross
• Agronomic and molecular characterization
• Phenotyping for AC, GC, GT
Activity
1:
Activity
2:
Activity3:
Schematic diagram of the methods used in the study
• Anther culture
Varietaldevelopment
•Selection of suitable parents
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Procedure followed in Activity 1
Rice cultivars (24)
• 164 SSRs
Genetic diversity analysis
Polymerase Chain Reactions
Electrophoresis
Staining of gels
Gel documentation
DNA extraction Anther culture
Selection ofappropriate parents
•Genetic similarities: NTSYS
•Cluster analysis: UPGMA
•Polymorphism InformationContent
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Variety Group Variety Group
Azucena 328 Trop. japonica IR65 Indica Basmati 370 Indica IR72 Indica
Milagrosa Indica IR74 Indica
Poloy tinawon Trop. japonica Bordagol 81726 Indica
Ifugao rice Trop. japonica PSBRc10 Indica
Nippon-bare Japonica PSBRc52 Indica
Pare baine pulut Trop. japonica PSBRc60 Indica
Dinorado Trop. japonica Burdagol Indica
IR29 Indica IR67406 Indica
IR841-85-1-1-2 Indica AR32-19-3-3 Indica
Azucena 47125 Trop. japonica AR32-19-3-4 Indica
Reket penjalin Trop. japonica Membrano Indica
The 24 quality rice cultivars evaluated for anther cultureresponse and genetic diversity
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RESULTS
SSR polymorphisms
• 92% polymorphism in SSR markers (151/164)
• 890 alleles detected in 24 genotypes
• Alleles ranged from 2 (RM114, RM312, RM408,RM420, RM451, RM556) to 17 (RM473B)
• Average of 5.89 alleles per locus
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• Average PIC value was 0.68 per
marker
• PIC ranged from 0.18 (RM420) to 0.91
(RM473B)
• Overall genetic diversity usingShannon’s diversity index was 0.71
PIC and Genetic diversity
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Dendogram of 24 rice genotypes derived from UPGMA clusteranalysis using Jaccard coefficient based on 151 polymorphicSSR markers
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Selected cultivars to be used as parents in the developmentof crosses
Cultivar GroupKey quality traits % GP *
AC GC GTBasmati 370 Indica L Soft L 23.3
Milagrosa Indica L Soft L 14.3
IR29 Indica L Soft L 7.7
IR72 Indica H Hard H 9.1IR74 Indica L Soft L 20.0
PSB Rc10 Indica H Hard H 12.0
PSB Rc60 Indica H Hard H 10.8
Nipponbare Japonica L Soft L 58.8Poloy tinawon Trop. japonica L Soft I 31.7
Azucena Trop. japonica I Medium I 33.3
Pare baine pulut Trop. japonica L Soft L 32.1
*Reckoned from the total number of induced calli
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•Anther culture
Albino plants
•Cytological evaluation in
actively dividing root-tip cells
Sterile plantsDoubled haploid(DH) plants
• Agro-morphological evaluation
•Molecular analysis (107/148 SSR)
Mapping population
Procedure followed in Activity 2
Development of crosses
F1sPSB Rc10 X Nipponbare
(selected parents)
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Crosses developed1. PSB Rc10 X Nipponbare2. IR29 X PSB Rc10
3. Basmati 370 X PSB Rc104. PSB Rc60 X Azucena5. IR72 X Milagrosa6. IR72 X Nipponbare7. Azucena x Poloy tinawon8. IR74 X Pare baine pulut
RESULTS
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PSB Rc10
Nipponbare
Phenotypic distribution of the 6 agronomic traitsof the AC-derived DH lines
F l f i i i DH li b (PSB
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F values for testing variations among DH lines, between parents (PSBRc10 and Nipponbare), and between DH lines and the parents for thedifferent agronomic traits under replicated trial.
Source ofVariation
F values1
PH (cm) PN TN PL (cm) % SF DTH2
Replication
Genotype
DH linesParents
DH vs P
Sampling error
2.36 ns
76.54 **
77.33 **91.37 **
4.83 *
<1
3.65 *
9.49 **
9.50 **16.41 **
2.08 ns
<1
0.34 ns
7.59 **
7.62 **11.77 *
1.87 ns
<1
0.16 ns
35.56 **
35.61 **66.85 **
0.55 ns
<1
0.57 ns
10.50 **
10.60 **3.98 *
10.29 *
<1
16.99 **
51.65 **
49.64 **98.50 **
68.21 **
CV (%) 5.4 14.9 15.9 11.31 8.0 2.6
1Ratio of the mean squares between each source of variation and the experimentalerror; ns = not significant; * P<0.05; ** P<0.00012No sampling error since observation was taken on a plot basis
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F values for variances within DH lines in comparison withthe checks for five agronomic traits.
Traits F value1
Significance ofdifference
Plant height (cm)
Tiller number
Panicle number
Panicle length (cm)
% Seed fertility
1.46
1.48
0.93
1.27
1.54
NS
NS
NS
NS
NS
1Ratio of an average of within DH line variance versus pooled variance ofthe parents (checks)
NS = not significant
A fi ld i f DH l t h i (A) i ti i i
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A field view of DH plants showing (A) variations in agronomictraits between lines and (B) uniformity within line (a closerlook of 4 DH lines)
A
B
DH 1 DH 2 DH 3
DH 4
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Polymorphism survey and distribution ofindica and japonica alleles
Polymorphism between parents = 72.3%(107/148)
Number of polymorphic markers/chromosomevaried from 4 (Chr 9) to 16 (Chr 8)
49.6% PSB Rc10 type, 48.2% Nipponbare type
99.84% of the total marker loci (14,243)
homozygous
Absence of non- parental or polymorphicbands in the DH populations
AC d i d DH li h f ith f th
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AC-derived DH lines homozygous for either of thealleles of parents at the RM 280 locus
P d f ll d i A i i 3
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Procedure followed in Activity 3
DH lines (219)
Seeds Leaf samples
•Grain quality
analyses
Phenotypic data(AC, GC, GT)
Traitscorrelation
DNA
• DNA extraction
• Molecular analysis 205 SSR markers
Genotypic data
QTL mapping
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Amylose content (AC) (Juliano 1971)
Gel consistency (GC) (Cagampang et al. 1973)
• flow characteristic of milled rice gel in 0.2 M potassiumhydroxide (KOH) measured by the length (in mm) of thecold gel in the culture tube held horizontally
Gelatinization temperature (GT) (Little et al. 1958)
• estimated by the extent of alkali spreading of rawmilled rice in a weak alkali solution (1.7% KOH)
Protocols employed in grain quality analyses
Genetic linkage map constructed using
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Genetic linkage map constructed usingMapManagerQTX
Significance thresholds to declare QTL for AC GC and GT
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Significance thresholds to declare QTL for AC, GC, and GT
3.3
3.1
3.2
RESULTS
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Correlation coefficients among the three traits.
Trait AC GC ASV (GT)
Gel consistency
Alkali spreading value
-0.627**
0.208*
1.000
-0.176*
1.000
*Significant at the 0.05 probability level.**Significant at the 0.01 probability level.
RESULTS
Distribution of AC GC and GT (expressed in ASV) in the
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Distribution of AC, GC, and GT (expressed in ASV) in theDH population
GC value:25 – 40 mm (Hard)41 – 60 mm (Medium)61 – 100 mm (Soft)
AC value:10 – 20% (Low)20 – 25% (Intermediate)> 25% (High)
ASV value:2 – 3 (High GT)4 – 5 (Intermediate GT)6 – 7 (Low GT)
QTL for cooking and eating quality traits among 219 DH lines
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TraitLocusname
Chr1 Marker interval LOD2 R2 (%)3 Additiveeffect4
Amylosecontent
qAC6aqAC6bqAC6c
666
RM469-RM170RM170-RM190RM197-RM225
38.6062.3320.91
65.074.043.0
5.886.374.77
Gelconsistency
qGC2qGC6aqGC6b
qGC6cqGC8
266
68
RM71-RM2634RM469-RM170RM170-RM190
RM197-RM225RM350-RM342A
3.2014.4823.81
10.613.59
4.023.035.0
23.04.0
-4.58-11.44-14.15
-11.32-4.80
Alkalispreadingvalue
qGT2qGT6aqGT6b
qGT6cqGT6d
266
66
RM3294-RM6233RM469-RM170RM170-RM190
RM197-RM225RM7023-RM3330
3.4810.9519.39
3.4352.52
3.09.0
16.0
4.062
0.190.350.47
0.23-0.93
QTL for cooking and eating quality traits among 219 DH linesderived from a cross between PSB Rc10 and Nipponbare.
1Chromosome number2Logarithm of odds score3Proportion of the total phenotypic variance explained by QTL4Positive values are associated with an increasing effect from PSB Rc10 alleles and negative
values are associated with an increasing effect from Nipponbare alleles
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Location of the quantitative trait loci (QTL) for amylosecontent, gel consistency, and gelatinization temperature.
Procedure in varietal development utilizing DH lines
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Doubled haploid (DH) lines
QTL mapping
• Agronomic and molecular characterization
Procedure in varietal development utilizing DH lines
Mappingpopulation
Varietal development
•2009 WS, 2010 DS & WS Preliminary Yield Trial (PYT)
Selected 12 DH lines based on highyield, good quality traits, and early to
medium maturityWith MR to R to blast; MS to MR to
BPH and GLH
SUMMARY AND CONCLUSION
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SUMMARY AND CONCLUSION
PSB Rc 10 and Nipponbare identified and selected as donorparentals for grain quality improvement
Anther culture
• enhanced genetic diversity
• generated homozygous and stable DH lines to constitute amapping population within short period of time
• developed outstanding/improved lines with high yield,resistance to pest, and with good quality traits
• shorten the breeding cycle (to reach PYT) by 1.5 years or 3
seasons)
Molecular marker analysis showed sufficient geneticdiversity among the 24 rice cultivars that could be tapped in
breeding for quality improvement and other variousagronomic traits
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Identified linked markers to QTLs (RM170, RM190(Wx gene), RM3330, RM7023 (Alk gene)
Identified QTLs and SSRs may be used in MAS breedingfor quality and other agronomic traits
Identified 13 QTLs- 3 (AC) and 5 each (GC and GT)
• Major QTL for AC corresponded very well to the Wx locus
• Major QTL for GT detected on the same locus as thereported Alk in Chr 6
SSR analysis proved DH lines to be unique geneticresources for developing elite breeding lines, mapping QTLs,
for (future) genomic research
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