Mapping of Quantitative Trait Loci (QTL) for Grain Cooking and Eating Quality using a Doubled Haploid (DH)

Population from Anther Culture of Indica/Japonica Hybrids of Rice (Oryza sativa L.)

VICTORIA CHAVEZ LAPITAN Toshinori Abe, Edilberto D. Redoa, Darshan S. Brar

RICE IS LIFE!

Rice (Oryza sativa L.) is . . . Most important cereal crop in the world Staple food for >half of human population Provides > one fifth of the calories consumed worldwide

 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 most rice breeding programs

Key cooking and eating quality traits:
Amylose content (AC)
Gel consistency (GC)

Gelatinization temperature (GT)

Quality improvement thru conventional breeding is difficult due to quantitative inheritance DNA marker technology facilitates understanding of complex quantitative traits.
Doubled haploid (DH) lines excellent materials for genetic studies because of their homozygosity and uniformity

OBJECTIVES
To determine the genetic diversity among a set of rice cultivars To generate DH lines as mapping population from a diverse cross through anther culture;

To identify QTLs for AC, GC, and GT utilizing the DH population

To demonstrate the novel use of the DH population for breeding and genomics through molecular analysis

Schematic diagram of the methods used in the study

Activity 1:

Rice cultivars (different quality traits)

Anther culture
Genetic diversity analysis Selection of suitable parents

Activity 2:

Development of crosses Anther culture of F1s

Selection of best cross

Doubled haploid (DH) lines

Agronomic and molecular

Varietal development

characterization

Phenotyping for AC, GC, GT Activity 3:

QTL mapping

Procedure followed in Activity 1

Rice cultivars (24)
DNA extraction
164 SSRs

Anther culture

Polymerase Chain Reactions

Electrophoresis Staining of gels

Selection of appropriate parents

Genetic similarities: NTSYS Gel documentation

Cluster analysis: UPGMA

Polymorphism Information Content

Genetic diversity analysis

The 24 quality rice cultivars evaluated for anther culture response and genetic diversity
Variety Azucena 328 Basmati 370 Group Trop. japonica Indica Variety IR65 IR72 Group Indica Indica

Milagrosa
Poloy tinawon Ifugao rice

Indica
Trop. japonica Trop. japonica

IR74
Bordagol 81726 PSBRc10

Indica
Indica Indica

Nippon-bare
Pare baine pulut Dinorado IR29 IR841-85-1-1-2 Azucena 47125 Reket penjalin

Japonica
Trop. japonica Trop. japonica Indica Indica Trop. japonica Trop. japonica

PSBRc52
PSBRc60 Burdagol IR67406 AR32-19-3-3 AR32-19-3-4 Membrano

Indica
Indica Indica Indica Indica Indica Indica

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

PIC and Genetic diversity

Average PIC value was 0.68 per marker

PIC ranged from 0.18 (RM420) to 0.91 (RM473B) Overall genetic diversity using Shannons diversity index was 0.71

Dendogram of 24 rice genotypes derived from UPGMA cluster analysis using Jaccard coefficient based on 151 polymorphic SSR markers

Selected cultivars to be used as parents in the development of crosses

Cultivar
Basmati 370 Milagrosa IR29 IR72 IR74 PSB Rc10 PSB Rc60 Nipponbare

Group
Indica Indica Indica Indica Indica Indica Indica Japonica

Key quality traits AC

L L L H L H H L

% GP *
23.3 14.3 7.7 9.1 20.0 12.0 10.8 58.8

GC
Soft Soft Soft Hard Soft Hard Hard Soft

GT
L L L H L H H L

Poloy tinawon
Azucena

Trop. japonica
Trop. japonica

L
I L

Soft
Medium Soft

I
I L

31.7
33.3 32.1

Pare baine pulut Trop. japonica

*Reckoned from the total number of induced calli

Procedure followed in Activity 2

Development of crosses
PSB Rc10 X Nipponbare
(selected parents)

F1s
Anther culture

Doubled haploid (DH) plants

Albino plants

Sterile plants
Cytological evaluation in actively dividing root-tip cells

Agro-morphological evaluation Molecular analysis (107/148 SSR)

Mapping population

RESULTS
Crosses developed
1. PSB Rc10 X Nipponbare 2. IR29 X PSB Rc10 3. Basmati 370 X PSB Rc10 4. PSB Rc60 X Azucena 5. IR72 X Milagrosa 6. IR72 X Nipponbare 7. Azucena x Poloy tinawon 8. IR74 X Pare baine pulut

Phenotypic distribution of the 6 agronomic traits of the AC-derived DH lines

PSB Rc10 Nipponbare

F values for testing variations among DH lines, between parents (PSB Rc10 and Nipponbare), and between DH lines and the parents for the different agronomic traits under replicated trial.
Source of Variation Replication Genotype DH lines Parents DH vs P F values1

PH (cm)
2.36 ns 76.54 ** 77.33 ** 91.37 ** 4.83 *

PN
3.65 * 9.49 ** 9.50 ** 16.41 ** 2.08 ns

TN
0.34 ns 7.59 ** 7.62 ** 11.77 * 1.87 ns

PL (cm)
0.16 ns 35.56 ** 35.61 ** 66.85 ** 0.55 ns

% SF
0.57 ns 10.50 ** 10.60 ** 3.98 * 10.29 *

DTH2
16.99 ** 51.65 ** 49.64 ** 98.50 ** 68.21 **

Sampling error
CV (%)
1Ratio

<1
5.4

<1
14.9

<1
15.9

<1
11.31

<1
8.0 2.6

of the mean squares between each source of variation and the experimental error; ns = not significant; * P<0.05; ** P<0.0001 2No sampling error since observation was taken on a plot basis

F values for variances within DH lines in comparison with the checks for five agronomic traits. Traits F value1 Significance of difference

Plant height (cm)

Tiller number Panicle number

1.46
1.48 0.93

NS
NS NS

Panicle length (cm)

% Seed fertility
1Ratio

1.27
1.54

NS
NS

of an average of within DH line variance versus pooled variance of the parents (checks) NS = not significant

A field view of DH plants showing (A) variations in agronomic traits between lines and (B) uniformity within line (a closer look of 4 DH lines)

DH 4

DH 1

DH 2

DH 3

Polymorphism survey and distribution of indica and japonica alleles

Polymorphism between parents = 72.3% (107/148) 49.6% PSB Rc10 type, 48.2% Nipponbare type Number of polymorphic markers/chromosome varied from 4 (Chr 9) to 16 (Chr 8) 99.84% of the total marker loci (14,243) homozygous Absence of non- parental or polymorphic bands in the DH populations

AC-derived DH lines homozygous for either of the alleles of parents at the RM 280 locus

Procedure followed in Activity 3

DH lines (219)

Seeds
Grain quality analyses

Leaf samples
DNA extraction

Phenotypic data (AC, GC, GT)

DNA
Molecular analysis 205 SSR markers

Traits correlation

Genotypic data QTL mapping

Protocols employed in grain quality analyses

Amylose content (AC) (Juliano 1971) Gel consistency (GC) (Cagampang et al. 1973)
flow characteristic of milled rice gel in 0.2 M potassium hydroxide (KOH) measured by the length (in mm) of the cold gel in the culture tube held horizontally

Gelatinization temperature (GT) (Little et al. 1958)

estimated by the extent of alkali spreading of raw milled rice in a weak alkali solution (1.7% KOH)

Genetic linkage map constructed using MapManagerQTX

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.

Distribution of AC, GC, and GT (expressed in ASV) in the DH population

AC value: 10 20% (Low) 20 25% (Intermediate) > 25% (High) GC value: 25 40 mm (Hard) 41 60 mm (Medium) 61 100 mm (Soft)

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 derived from a cross between PSB Rc10 and Nipponbare.
Trait Amylose content Gel consistency Locus name qAC6a qAC6b qAC6c qGC2 qGC6a qGC6b qGC6c qGC8 qGT2 qGT6a qGT6b qGT6c qGT6d Chr1 6 6 6 2 6 6 6 8 2 6 6 6 6 Marker interval RM469-RM170 RM170-RM190 RM197-RM225 RM71-RM2634 RM469-RM170 RM170-RM190 RM197-RM225 RM350-RM342A RM3294-RM6233 RM469-RM170 RM170-RM190 RM197-RM225 RM7023-RM3330 LOD2 38.60 62.33 20.91 3.20 14.48 23.81 10.61 3.59 3.48 10.95 19.39 3.43 52.52 R2 (%)3 65.0 74.0 43.0 4.0 23.0 35.0 23.0 4.0 3.0 9.0 16.0 4.0 62 Additive effect4 5.88 6.37 4.77 -4.58 -11.44 -14.15 -11.32 -4.80 0.19 0.35 0.47 0.23 -0.93

Alkali spreading value

1Chromosome

number 2Logarithm of odds score 3Proportion of the total phenotypic variance explained by QTL 4Positive 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 amylose content, gel consistency, and gelatinization temperature.

Procedure in varietal development utilizing DH lines Doubled haploid (DH) lines

Agronomic and molecular characterization 2009 WS, 2010 DS & WS Preliminary Yield Trial (PYT)

Mapping population

Varietal development Selected 12 DH lines based on high yield, good quality traits, and early to medium maturity With MR to R to blast; MS to MR to BPH and GLH

QTL mapping

SUMMARY AND CONCLUSION

Molecular marker analysis showed sufficient genetic diversity among the 24 rice cultivars that could be tapped in breeding for quality improvement and other various agronomic traits PSB Rc 10 and Nipponbare identified and selected as donor parentals for grain quality improvement

Anther culture
generated homozygous and stable DH lines to constitute a mapping population within short period of time enhanced genetic diversity

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)

SSR analysis proved DH lines to be unique genetic resources for developing elite breeding lines, mapping QTLs, for (future) genomic research 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 the reported Alk in Chr 6 Identified linked markers to QTLs (RM170, RM190 (Wx gene), RM3330, RM7023 (Alk gene)

Identified QTLs and SSRs may be used in MAS breeding for quality and other agronomic traits