adss_oct26_mapping of quantitative trait loci

8/4/2019 ADSS_Oct26_Mapping of Quantitative Trait Loci

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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



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




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)




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




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




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




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




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




• 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




Dendogram of 24 rice genotypes derived from UPGMA clusteranalysis using Jaccard coefficient based on 151 polymorphicSSR markers




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




•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


Development of crosses

F1sPSB Rc10 X Nipponbare

(selected parents)




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




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




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




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




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




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




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





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




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




Genetic linkage map constructed usingMapManagerQTX

Significance thresholds to declare QTL for AC GC and GT




Significance thresholds to declare QTL for AC, GC, and GT

3.3

3.1

3.2

RESULTS




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




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




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




Location of the quantitative trait loci (QTL) for amylosecontent, gel consistency, and gelatinization temperature.

Procedure in varietal development utilizing DH lines




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




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




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


adss_oct26_mapping of quantitative trait loci

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