THESIS

THESIS 

REGULATION OF GENES CONTROLLING GRAIN 

ANTHOCYANIN AND PROANTHOCYANIDIN (CONDENSED 

TANNINS) ACCUMULATION IN RICE 

KIJANAN WIRIYASUK 

GRADUATE SCHOOL, KASETSART UNIVERSITY 

2005

THESIS 


ANTHOCYANIN AND PROANTHOCYANIDIN (CONDENSED 

TANNINS) ACCUMULATION IN RICE 

KIJANAN WIRIYASUK 

A Thesis Submitted in Partial Fulfillment of 

the Requirements for the Degree of 

Master of Science (Genetic Engineering) 

Graduate School, Kasetsart University 

2005 

ISBN 974-9834-79-8

ACKOWLEDGEMENTS 

This work was complete with a lot of help from a number of people. I would 

sincerely like to acknowledge to my committee members, Dr. Somvong Tragoonrung, 

Associate Professor Dr. Apichart Vanavichit, and Dr. Vipa Hongtrakul. I appreciated 

for their advice. 

Thank to National Center for Genetic Engineering and Biotechnology 

(BIOTEC), the two-year scholarship for my thesis. 

My thank also go to The Rice Gene Discovery Unit and The DNA 

Technology Unit for supporting my research facilities and thanks to their supportive 

and encouragement. 

Finally, for my family and my friends who always give me encouragement, I 

am thankful for all your help. 

Kijanan Wiriyasuk 

April, 2005

TABLE OF CONTENTS 

i 

Page 

TABLE OF CONTENTS…………………………………………………….. i 

LIST OF TABLES………………………………………………………….... iii 

LIST OF FIGURES…………………………………………………………... iv 

LIST OF ABBREVIATIONS………………………………………………… vi 

INTRODUCTION……………………………………………………………. 1 

LITETATURE REVIEWS…………………………………………………… 3 

Genetic of the anthocyanin pigmentation in rice……………………... 

Variation in tissue-specific distribution of anthocyanins 

3 

among rice lines………………………………………………………. 5 

Biochemical characterization of pigments……………………………. 5 

Flavonoid biosynthetic pathway in rice………………………………. 6 

Molecular isolation and characterization of rice cDNA clones………. 9 

The anthocyanin pathway in rice is ultraviolet light-responsive……… 

Molecular manipulation of the anthocyanin pathway to 

11 

improve disease resistance in rice…………………………………….. 12 

MATERIALS AND METHODS……………………………………………... 14 

Plant materials………………………………………………………… 14 

Phenotyping of grain anthocyanin and proanthocynidin 

content in rice…………………………………………………………. 14 

Plant DNA extraction…………………………………………………. 15 

Genes specific primer design and amplification……………………… 16 

Single-strand conformational polymorphism (SSCP)………………… 17 

RNA Analyses………………………………………………………… 

Analysis nucleotide sequencing of anthocyanin 

17 

biosynthetic genes…………………………………………………….. 17 

Place and Duration……………………………………………………. 18

TABLE OF CONTENS (Continued) 

ii 

Page 

RESULTS…………………………………………………………………….. 19 

Isolation of seed color mutants……………………………………….. 19 

Quantitative of Anothocyanin………………………………………… 21 

Anthocyanin accumulation during grain development……………….. 22 

Regulatory Anthocyanin and Proanthocyanidin genes……………….. 23 

Defining region of rice seed color……………………………. 23 

Genomic organization of OSB1 and OSB2…………………... 25 

2-bp deletion in OSB1 generated a frame shift………………. 25 

Temperature-sensitive DFR transcript……………………………….. 28 

Genomic organization of DFR……………………………….. 28 

Temperature directing transcription profiles of DFR………… 29 

Post-transcriptional regulation of DFR is still a black box….. 30 

ANS, a key reaction for coloring in anthocyanin biosynthesis………. 32 

DISSCUSSION…………………………………………………………….... 36 

CONCLUSSION…………………………………………………………….. 40 

LITERATURE CITED………………………………………………………. 41 

APPENDIX…………………………………………………………………... 46

LIST OF TABLES 

Table Page 

1. The anthocyanin gene-pigment system and phenotypic 

effects on rice…………………………………………………………… 4 

2. Anthocyanin content of the rice varieties……………………………… 22 

iii

LIST OF FIGURES 

Figure Page 

1. The major anthocyanidin pigment in rice was indentified 

as cyanidin and the minor one as peonidin…………………………. 7 

2. Diagram of anthocyanin-producing cell in rice…………………….. 8 

3. Mutation of JHN seed color (BW1-4)…………………………….... 19 

4. Phylogenetic tree of rice variety using the NTSYSpc 2.10……….... 20 

5. Varying extractability of rice anthocyanin pingmentation…………. 22 

6. Anthocyanin accumulation during grain development 

in JHN purple rice………………………………………………….... 23 

7. Location of gene controlling grain color……………………………. 24 

8. The structure of OSB1 and OSB2 genes…………………………… 26 

9. Sequencing of OSB1-A fragment contrained 2-bp deletion………... 27 

10. Amplifed product OSB1-A fragment run SSCP on 

8% acylamide gel…………………………………………………… 28 

11. Structure of rice dihydroxyflavanol-4 reductase (DFR) 

gene including primer positions…………………………………….. 29 

12. Expression of DFR determined by RT-PCR revealed 

transcription profiles in summer and winter………………………... 30 

13. Two alleles of DFR, DFR x and DFR y .DFR x is 

temperature-sensitive allelethat found in deeppurple 

grain (a, b). DFR y is the unspliced allele found in 

normal white rice (c)……………………………………………….. 32 

14. Structure of rice anthocyanidin synthase (ANS) gene 

including primer positions………………………………………….. 33 

15. PCR-SSCP method detecting anthocyanin biosynthetic 

genes mutation……………………………………………………… 33 

16. DNA sequence alignment of ANS3 from different 

rice strains……………………………………………………….….. . 34 

iv

LIST OF FIGURES (Continued) 

Figure Page 

17. Cladogram sequencing of ANS3 fragment…………………………. 35 

18. Comparison between REB1 domain of C1 Nipponbare 

with C1 Purpleputu. REB1…………………………………….….. 37 

19. Mechanism of anthocyanin formation, leucoanthocyanidin 

to anthocyanidin 3-glucoside, catalyzed by ANS and 3-GT, 

and transport to vacuoleds………………………………………….. 39 

v

LIST OF ABBREVIATIONS 

ANS = Anthocyanidin synthase gene 

BAC = Bacterial Artificial Chromosome 

BW1-4 = Jao Hom Nin Mutant 1-4 

C1 = Colored-1 gene 

cDNA = Complementary 

DFR = Dihydroflavonol 4-reductase gene 

DH = Double Haploid 

g = gram 

JHN = Jao Hom Nin 

Kb = Kilobase 

KDML105 = Khao Dawk Mali 105 

ml = Milliter 

mM = Millimolar 

mRNA = Messenger Ribonucleic Acicd 

NCBI = National Center for Biotechnology Information 

ng = Nanogram 

nm = nanometer 

ORF = Open Reading Frame 

OSB1 = Oryza sativa. Booster1 gene 

OSB2 = Oryza sativa Booster 2 gene 

PCR = Polymerase Chain Reaction 

QTL = Quantitative Trait Locus 

RGP = Genome Research Program 

RT-PCR = Reverse-transcribed Polymerase Chain Reaction 

SSCP = Single Stand Conformation Polymorphism 

U = Unit 

µL = Microliter 

µM = Micromolar 

°C = Degree Celsius 

vi


ANTHOCYANIN AND PROANTHOCYANIDIN 

(CONDENSED TANNINS) ACCUMULATION IN RICE 

INTRODUCTION 

Rice (Oryza sativa L.) is one of the most important crop in Thailand and is the 

main source of energy, vitamin and protein and the top agricultural product being 

exported world wide. People are more concern about health, thus the vision regarding 

rice consumption was improved. This is the reason, why the brown rice is more 

preferable than before because rice meal contains high nutritional value such as 

protein, oil and cellulose. Color is one interesting character of brown rice that is 

present in the pericarp. Three different colors including light-brown, red and purple 

are the main colors that are produced by flavanoid biosynthesis. Flavonoids are 

secondary metabolites that are unique to higher plants. They are well known for the 

red, purple, and brown pigmentation they give to flowers, fruit, and grain. Flavonoids 

fulfill numerous physiological functions during plant life and also serve as beneficial 

micronutrients in human and animal diets (reviewed by Koes et al., 1994; Shirley, 

1996; Mol et al., 1998; Harborne and Williams, 2000). Rice contains three major 

classes of flavonoids: the anthocyanins (red to purple pigments), the flavonols 

(colorless to pale yellow pigments), and the proanthocyanidins (colorless pigments 

that turn to brown), which also are known as condensed tannins. Anthocyanins and 

flavonols are synthesized in vegetative parts, whereas flavonols and 

proanthocyanidins accumulate in pericarp (Reddy et al., 1995). Color of rice is not 

only attractive for consumer, it also appear to be antioxidant which is the important 

mechanism to protect consumers from body disorder (Canada et al., 1990; Myara et 

al., 1993). Hydrolyzed anthocyanin from red rice was effective on the suppression of 

tumor growth (Koide et al., 1996). In the future, health care providers may hand out 

proanthocyanidin pills as readily as they recommend aspirin today. A steady stream of 

animal and in vitro studies supplemented by epidemiological evidence and a 

1

smattering of preliminary human studies reveal numerous health benefits associated 

with these compounds. Chief among the benefits is antioxidant protection against 

heart disease and cancer. During the last decade, rapid advances of molecular genetic 

including morphological, isozyme and DNA markers used as genetic marker for 

construction of linkage maps (Yoshimura et al., 1997). Advance molecular 

technology provides us with information and tools useful in locating marker related to 

anthocyanin and proanthocyanidin accumulation in rice pericarp through QTL 

mapping approach and study RT-PCR of regulatory gene controlling grain 

anthocyanin and proanthocyanidin content in rice during grain development which 

will understranding the genetic base is of anthocyanin and proanthocyaidin 

pigmentation that rice breeder can use improve qualities of rice grain color and 

develop to regulatory anthocyanin gene as reporters in rice transformation. 

The objectives of this study are: 

1. To determine total anthocyanin and proanthocyanidin content among 

different rice varieties. 

2. To utilize the linkage map using F2 population derived from a cross 

between KDML105 and Jao Hom Nin in order to locate the QTL position of the traits 

related to anthocyanin and proanthocyanidin content in rice. 

3. To clone anthocyanin biosynthetic gene and detect gene mutation. 

4. To investigate regulation of genes controlling grain anthocyanin and 

proanthocyanidin accumulation in rice using RT-PCR. 

2

LITERATURE REVIEW 

Genetic of the anthocyanin pigmentation in rice 

Anthocyanin pigmentation in rice plant has been extensively reported by 

earlier breeders and geneticists (Nagao et al., 1962; Kadaw, 1974; Takahashi, 1982; 

Maekawa and Kita, 1987; Kinoshita and Takahashi, 1991; Reddy, 1996; Reddy et al., 

1994, 1995, 1997, 1998). Genetic analyses were mainly concentrated on phenotypic 

description, identification of specific loci, and chomosomal map position. Broadly, 

the anthocyanin gene pigment system and its phenotypic variation in rice consist of 

structural gene, the C (Chromogen), A (Activator) and the Rc and Rd (determining the 

brown pericarp). The regulatory genes P (Purple) and Pl (Purple leaf), each with a 

number of alleles, govern the distribution of purple pigments in various plant organs. 

A collation of known genes of rice, their phenotypic effects, and interaction patterns 

is presented briefly in Table 1. However, noting is known about specific gene 

products and enzymes controlling individual biosynthetic step reactions and the 

associated regulatory circuits of the pathway. The pattern of anthocyanin 

pigmentation in the rice plant is thus determined mainly by the allelic status of 

individual genes and complex interactions between them. Intensity and shades of 

color are marginally influenced by nongenetic factor such soil condition, mineral, 

nutrition, pH, temperature, and light. 

3

Table 1 The anthocyanin gene-pigment system and phenotypic effects on rice. 

Gene Phenotypic effect 

Structural 

C (chromogen) 

A (activator) 

Rc (brown pericarp) 

Rd (brown pericarp) 

Regulatory 

P (purple) 

Pl (purple leaf) 

Pn (purple node) 

Prp (purple pericarp) 

Responsible for anthocyanin production: with an allelic 

series of C B , C Br , C + (null), etc. 

Activation of C gene; essential for anthocyanin: with an 

allelic series of A S , A E , A, A + (null), etc. 

Synthesis of pigments in pericarp 

Synthesis of pigments in pericarp 

Distributor of anthocyanin pigments in the apiculus: alleles 

P, P K , P + (null), etc. 

Localizer of anthocyanin in leaf: alleles Pl w (leaf blade, 

leaf sheath, auricles, ligule, and pericarp); Pl (leaf blade, 

leaf sheath, collar, auricles, ligule, node, and internode); 

Pl i (leaf blade, leaf sheath, ligule, and internode); Pl + (null 

allele resulting into color less phenotype of tissue). 

Localizer of anthocyanin in the node 

Localizer of anthocyanin in the pericarp 

Inhibitory 

Dominant inhibitor to purple anthocyanin 

I-Pl (inhibitor of 

purple leaf) 

Inhibit action of both Pl 

I-Pl1, I-Pl2, I-Pl3 

I-Pl4, I-Pl5 

I-Pl6 

Ilb (inhibitor of 

purple leaf) 

w and Pl i alleles 

Inhibit action of the Prp locus 

Inhibits action of Pl i allele 

Inhibits leaf blade pigmentation 

Sources: Chang and Jordan (1963), Takahashi (1982), Kinoshita and Takahashi (1991); 

adapted from Reddy et al (1995). 

4

Variation in tissue-specific distribution of anthocyanins among rice lines 

There is significant variation, both qualitative and quantitative, among rice 

varieties in the display of red/purple color phenotypes in various plant organs such as 

leaf, stem, node, internode, auricle, ligule, leaf sheath, stigma, apiculus, and pericarp. 

The distribution of red/purple color in different plant parts among indica lines showed 

considerable variation (Reddy et al., 1995). Some lines show color in almost all aerial 

tissues, whereas some show no red/purple color in any tissues. A few others exhibit an 

intermediate phenotype with color in various parts and finally, certain lines show no 

anthocyanin color in aerial parts but exhibit brown color in pericarp tissue (Reddy et 

al., 1995). The genotype of the above selected lines is predicted and then classfied, 

based on the pigment variation among well-defined japonica lines. These lines served 

as a base material for isolation of mutants and subsequently, the genes of pathway. 

Biochemical characterization of pigments 

Rice plants accumulate mainly two anthocyanin pigments, cyanidin and 

peonidin (Figure 1), an o-methyl derivative of cyanidin. This was confirmed by a 

variety of standard techniques: thin layer chromatography, proton-NMR spectroscopy, 

UV-VIS spectroscopy, and standard organic methods and cochromatography with 

authentic compounds (Reddy et al., 1995). Incidentally, this combination of 

anthocyanin pigment appears to be exclusive to indica rice. In certain japonica plants, 

the presence of malvidin was reported (Takahashi, 1957). In addition, rice plant seem 

to accumulate a unique class of pigments called proanthocyanins, imparting brown 

color, particularly in the pericarp of N22B. These pigments yielded cyanidin and 

peonidin on hydrolysis (Reddy et al., 1995). 

Qualitative and quantitative analyses of pigment extracts revealed that floral 

derived tissue accumulates as much as five times more pigments than vegetative 

tissue with about a tenfold increase in peonidin content in the apiculus, hull, and 

pericarp. On the other hand, vegetative tissues have cyanidin as the major pigment 

with very little of peonidin. Thus, the observed pigmentation pattern in a given tissue 

5

in rice reflects complex nonallelic gene interactions and the role of tissue-specific 

regulatory mechanisms. The assorted color shades from brown to red to deep purple 

could be due to different chemical modifications such as hydroxylation, glycosylation, 

methylation, acylation, and polymerization of the basic anthocyanidin molecule, apart 

from the effects of pH and the presence of copigments (Holton et al., 1993). For 

instance, methylation appears to be predominant in floral-derived tissue, being at its 

maximum in the pericarp of Purpleputtu (PP) rice. In as much as the pigment analyses 

were with anthocyanidin aglycones, it was not possible to assess the native glycosidic 

nature of these pigments. Also, the composition of flavonoids in rice has yet to be 

established. 

Flavonoid biosynthetic pathway in rice 

The flavonoid biosynthetic pathway in rice is well established (Reddy et al., 

1995). A generalized flavonoid biosynthetic pathway is shown in Figure 2. The 

precursors for the synthesis of all flavonoids, including anthocyanins, are malonyl- 

CoA and p-coumaroyl-CoA. Chalcone synthase (CHS) catalyzes the stepwise 

condensation of three acetate units from malonyl-CoA with 4-coumaroyl-CoA to 

yield chalcononaringenin. Chalcone isomerase (CHI) then catalyzes the stereospecific 

isomerization of the yellow-colored chalcononaringenin to the colorless naringenin 

(NAR). NAR is converted to dihydrokaempferol (DHK) by flavonone 3-hydroxylase 

(F3H) or converted to eriodictyol (ERI) by flavonoid 3-hydroxylase (F3’H). DHK can 

subsequencetly be hydroxylated by flavonoid 3-hydroxylase (F3’H) or ERI 

hydroxylated by flavonone 3-hydroxylase (F3H) to produce dihydroquercetin (DHQ). 

DHQ is reduced to leucocyanidin (leucoanthocyanidin) by dihydroflavonol 4reductase 

(DFR). Leucoanthocyanidinreductase (LAR) which converts leucocyanidin 

to catechin, thereby initiating the polymerisation into procyanidin (brown). 

Anthocyanidin syntase (ANS), convert leucocyanindid to cyanidin (red) when is 

glycosylated to cyanidin-3-o-glucoside by flavonoid 3-glucosyltransferase (FGT) and 

peonidin-3-o-glucoside which is methylated by anthocyanin methyl transferase 

(AMT) that transferred to vacuole by glutathione s-transferase (GTS). 

6

Cyanidin Peonidin 

Procyanidin 

Figure 1 The major anthocyanidin pigment in rice was indentified as cyanidin and 

the minor one as peonidin. The major proanthocyanidin pigment in rice 

was tentatively identified as procyanidin. 

7

Figure 2 Diagram of anthocyanin-producing cell in rice. Regulatory genes for 

anthocyanin pigmentation pathway that activate their expression: C1 

(colored-1) and R (red). Inhibitory genes are I-PL (inhibitor of purple leaf) 

and I-LB (inhibitor of leaf blande) that inhibitory activity.Genes are 

represented in enzyme names are abbreviated as follows: phenylalanine 

ammonia lyase (PAL), cinamate-4-hydroxylase (C4H), 4-Coumarate : CoA 

ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), 

flavanone 3-hydroxylase (F3H), flavonoid 3’ hydroxylase (F3’H), 

dihydroflavonol 4-reductase (DFR), flavonol systhase (FLS), 

leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), 

flavonoid 3-glucosyltransferase (3GT), anthocyanin methyl transferase 

(AMT), glutathione s-transferase (GTS) and data not available (NA). 

Indicates Regulation. Indicates Inhibition. Used in floral 

modification (Reddy et al., 1995, 1996; Madhuri et al., 1999). 

8

Molecular isolation and characterization of rice cDNA clones 

Structural genes 

Chalcone synthase (CHS) 

A cDNA library was made from poly A + mRNA from young 

developing leaves of Purpleputtu (PP) in λgt NM 1149-Pop13 cells using standard 

protocols. This library was screened and several cDNA clones were isolated. Using 

maize Zm:CHS:C2, the maize C2 cDNA encoding chalcone synthase as a probe, a 

cDNA clone, OS-CHS, was isolated. This cDNA clone was further characterized and 

sequenced (Scheffler et al., 1995). The sequence comparison of OS-CHS with 

Zm:C2:CHS revealed 86.3% homology, and with Zm:Whp:CHS, 85.4% homology 

within the translated region. Western analysis using Zm:CHS antisera also leads to the 

same conclusion. Further, these proteins are of comparable size. The OS-CHS 

sequence was mapped to chromosome 11 of rice using a restriction fragment length 

polymorphism mapping strategy (Reddy et al., 1996c). Chalcone synthase catalyses 

the formation of naringenin chalcone, the first flavonoid carbon-15 skeleton, which is 

a condensation product of three molecules of malonyl CoA and one molecule of 4coumaroyl 

CoA. 

Dihydroflavonol 4-reductase (DFR) 

A cDNA clone to the Zm:A1 probe was isolated after in vivo 

excision in the form of pBluescript SK(-) from λZAP cDNA library derived from 

mRNA isolated from UV-B containing white light treated 4-day-old etiolated 

Purpleputtu (Oryza sativa L.) seedings (Reddy et al., 1996b). Dihydroflavonol 4reductase 

catalyses the NADPH-dependent conversion of dihydroquercetin into 

unstable corresponding leucoanthocyanidins, the immediate precursors for the 

anthocyanins. 

9

Anthocyanidin synthase (ANS) 

A cDNA clone hybridizable to the Zm:A2 probe was isolate 

and partially sequenced. The sequence comparison between Os:Ans and Zm:A2 at the 

DNA and protein (deducedfrom cDNA) levels revealed an extensive homology (data 

not presented). The a2 mutant of maize is known to cause a genetic block in the 

anthocyanin pathway leading to accumulation of colorless leucoanthocyanidin (Coe et 

al., 1988). The ans mutant, therefore, in principle, should either accumulate 

leucoanthocyanidin or proanthocyanidin. In fact, the mutant lines N22B and G962 

accumulate detectable amounts of proanthocyanidins and leucoanthocyanidins, 

respectively (Reddy et al., 1995), and therefore are potential ans mutants. 

Regulatory genes 

Booster gene (B) 

The Purple leaf (Pl) locus of rice (Oryza sativaL.) affects 

regulation of anthocyanin biosynthesis in various plant tissues. The tissue-specific 

patterns of anthocyanin pigmentation, together with the syntenic relationship, indicate 

that the rice Pl locus may play a role in the anthocyanin pathway similar to the maize 

R/B loci. Sakamoto et al. (2001) isolated two cDNAs showing significant identity to 

the basic helix-loop-helix (bHLH) proteins found in the maize R gene family. OSB1 

appeared to be allelic to the previously isolated R homologue, Ra1, but showed a 

striking difference at the C-terminus because of a 2-bp deletion. Characterization of 

the corresponding genomic region revealed that the sequence identical to a 5′-portion 

of OSB2 existed ~10-kb downstream of the OSB1 coding region. OSB2 lacks a 

conserved C-terminal domain. A transient complementation assay showed that the 

anthocyanin pathway is inducible by OSB1or OSB2. These results suggest that the Pl w 

allele may be complex and composed of at least two genes encoding bHLH proteins. 

10

Colored-1 gene (C1) 

The C locus is located on the short arm of chromosome 6 and is 

linked to the wx which also shows allelic differentiation among rice cultivars. Based 

on synteny of maps between rice and maize, the region including these genes is 

present on chromosome 9 of maize and the maize C1 anthocyanin regulatory gene is 

located in the region. The C1 encodes a MYB like protein factor which activates the 

transcription of a number of structural genes involved in anthocyanin pigment 

biosynthetic pathway (Paz Ares et al., 1987). The rice homologue (Os-C1) of the 

maize C1 was cloned from cDNA library of Purpleputtu seedlings (Reddy et al., 

1998). 

The anthocyanin pathway in rice is ultraviolet light-responsive 

The fact that the anthocyanin pathway in rice is ultraviolet (UV) lightresponsive 

(Reddy et al., 1994) is significant in view of the continuous depletion of 

the ozone layer in the atmosphere and the resulting increase in the incidence of UV 

light causing damage to plant life. Biochemical analysis revealed that this UV lightresponsive 

anthocyanin synthesis appears to be mediated by a specific phase of 

phenylalanine ammonia lyase activity (Reddy et al., 1994). Preliminary analysis 

suggests that rice seedlings seem to have a specific UV-B receptor in addition to 

general photoreceptor, the phytochrome. The UV-B-induced anthocyanin biosynthesis 

precedes the activation of genes of the anthocyanin pathway. Enzyme analyses 

revealed that phenylalanine ammonia lyase (Reddy et al., 1994), CHS, and F3GT 

showed enhanced activities under UV-B light. Northern analysis also substantiated 

these results. In addition, expression of the putative Gst gene (encoding glutathione-Stransferase) 

has also been shown to be inducible by UV-B in rice seedlings (Madhuri 

et al., 1994). 

A cDNA library from poly A+ mRNA of UV-B light-induced leaves was 

constructed and screened for genes of the anthocyanin pathway. This library would be 

11

a source of not only the anthocyanin genes, but also of UV-B responsive elements. 

Thus, the anthocyanin pathway could serve as a model system to study the genetic 

basis of response to light, particularly UV-B, and molecular mechanisms of signal 

transduction. 

Molecular manipulation of the anthocyanin pathway to improve disease 

resistance in rice 

Flavonoids as plant defense molecules 

The antibacterial and antifungal properties of flavonoids are well 

documented. Lamb et al. (1989) reported that flavonoids play a role in conferring 

disease resistance in many plants. Proanthocyanidins (Scalbert, 1991) and 3deoxyanthocyanidins 

(Snyder et al., 1991) have been shown to accumulate when 

plants are infected and are believed to be defense compounds. However, to date, 

direct evidence for such protective roles of these compounds is still lacking. Hence, 

we are looking at the toxic effects of flavonoids against major rice pathogens such as 

Xanthomonas oryzae pv. oryzae (Xoo; bacterial leaf blight), Pyricularia oryzae 

(blast), and Rhizoctonia solani (sheath blight). Some purified flavanones, flavonols, 

and phenylpropanoids were screened against the rice pathogens in an in vitro toxicity 

assay. The reaction response was highly varied where the tested compounds caused 

significant growth inhibition of the pathogens at micromolar concentrations. 

Naringenin (the first flavonoid intermediate committed to the anthocyanin pathway) 

showed a broad spectrum inhibition to six strains of Xoo tested. Liquid culture assays 

with naringenin also showed a tenfold reduction in the growth of Xoo after a 12-h 

shaker incubation at 28 o C. However, none of the compounds had any significant 

effect on the mycelial inhibition of P. oryzae or R. solani (Reddy, 1996). Attempts are 

under way to study the response of pigmented and nonpigmented rice cultivars, under 

blast and blight infection, by determining the flavonoid profiles, levels of 

phenylpropanoid and flavonoid pathway enzymes, and specific transcripts Role of 

flavonoids in disease resistance The present trend in developing defense strategies in 

12

many plants is to manipulate the response of defense genes to pathogen attack or to 

develop transgenic plants with engineered genes that inhibit the pathogen. A possible 

alternative strategy is to enhance the endogenous levels of selected phenylpropanoids 

and flavonoids by transferring the appropriate regulatory genes into rice plants. 

Enhanced accumulation of specific intermediates can also be achieved by using a 

combination of overexpression and antisense strategies. Such “smart plants” should 

hyper-accumulate flavonoid intermediates of interest and thereby should have their 

resistance to pathogen attack enhanced. Efforts are under way to make a complete 

repertoire of transcriptional fusion constructs of specific anthocyanin genes for 

overexpression and also inhibition, by antisense sequences, of the anthocyanin 

pathway. Such constructs are made for almost all the required structural and 

regulatory genes of the pathway. These constructs will enable us, through a transgenic 

approach, to test the premise that flavonoids play a role in disease resistance. Thus, 

the manipulation of the pathway and its eventual use to generate transgenics with 

improved disease resistance would ultimately prove to be a powerful technique. In 

addition, these gene constructs can be used possibly to alter the shades of color and 

intensity, tissue-specific distribution, and the composition of the responsible pigments 

in the variety of plant species, including ornamentals. 

13

1. Plant materials 

MATERIALS AND METHODS 

Rice strains used in this study were as follows: Oryza sativa strains Jao Hom 

Nin (JHN), Khao Dawk Mali 105 (KDML105) and Jao Hom Nin mutant (BW1-4), 

provided by Center of Excellence for Rice Molecular Breeding and Product 

Development, National Center for Agricultural Biotechnology, Kasetsart University, 

Kamphangsaen, Nakorn Pathom, Thailand. 

2. Phenotyping of grain anthocyanin and proanthocynidin content in rice 

2.1. Anthocyanin content 

On the basis of extractability results, a simple, rapid method for 

determining total anthocyanin in pigmented rice was established (Abedel and Hucl, 

1999). A ground rice sample (3 g) was weighed in a 50-ml centrifuge tube, and 24 ml 

of acidified ethanol (ethanol and HCl 1.0N, 85:15,v/v) was added. The solution was 

mixed and adjusted to pH 1 with 4N HCl. The resulting solution was shaken for 15 

min, and was readjusted to pH 1 if necessary, and the solution was shaken for an 

additional 15 min. The tube was centrifuged at 12,000 rpm for 30 min, and the 

supernatant was poured into a 50-ml volumetric flask and made up to volume with 

acidified ethanol. Absorbance was measured at 535 nm against a reagent blank. 

Cyanidin 3-glucoside or Kuromanin from Extrasynthese (Genay, France) was used as 

a standard pigment. A series of Cyanidin 3-glucoside standard solutions was prepared 

at 0-0.02 mmol (0-27 ug/3 ml). Absorbance was read at 535 nm against a reagent 

blank. The concentrations showed a linear relationship against absorbance and had 

regression and determination coefficients of 0.0197 and 0.999, respectively. Total 

anthocyanin content per sample (mg/kg) was calculated as cyanidin 3-glucoside: 

C = (A/ε) x (vol/1,000) x MW x (1/sample wt) x 10 6 

14

Where C is concentration of total anthocyanin (mg/kg), A is absorbance 

reading, ε is molar absorptivity (cyanidin 3-glucoside = 25,965 cm -1 M -1 ), vol is total 

volume of anthocyanin extract, and MW is moleculer weight of cyanidin 3-glucoside 

= 449. Under test conditions, the equation formula can be simplified to: 

C = (A/25,965) x (50/1,000) x 449 x (1/3) x 10 6 

or C = A x 288.21 mg/kg 

Beer’ law was used to calculate molar absorptivity of cyanidin 3-glucoside, which 

ranged from 25,591 cm -1 M -1 to 26,559 cm -1 M -1 , with an average of 25,965 cm -1 M -1 , 

standard deviation of 520, and coefficient of variation of 2%. Mean molar 

absorptivity was used to calculate the concentration of total anthocyanins in 

pigmented rice samples. 

2.2. Proanthocyanidin content 

Total proanthocyanidin in rice was established (Reddy et al., 1995). A 

ground rice seed (3 g) were extracted with 10% aqueous methanol (1ml/50mg) for 24 

h at room temperature with occasional shaking. To 6 ml of the pooled extract, 4.5 ml 

of water and 12 ml of chloroform were added. The resulting solution was shaken for 

15 min. The tube was centrifuged at 12,000 rpm for 30 min, and the supernatant was 

poured into a 50-ml volumetric flask and made up to volume with 10% aqueous 

methanol. Absorbance was measured at 457 nm against a reagent blank. 

3. Plant DNA extraction 

Freshly collected leaf tissue was ground into fine powder using liquid nitrogen 

with a mortar and pestle. Twenty ml of 1.5x CTAB extraction buffer, preheated at 65˚ 

C was added in 40 ml tube containing the ground tissue. The buffer tissue mixture 

was gently mixed to ensure even dispersal of the plant material in the buffer and was 

incubated at 65˚C for 1 hour with occasional swirling. The mixture was cooled at 

room temperature and equal volume of chloroform/isoamyl alcohol (24:1) was added. 

The tube were inverted repeatedly but gently and were centrifuged at 4000 rpm for15 

15

min at room temperature. The upper layer was transferred into a new centrifuge tube 

and 1 ml of 10x CTAB was added. Equal volume of chloroform/isoamyl alcohol was 

again added for the second round extraction of carbohydrates and other debris. The 

mixture was centrifuged with the same condition and the aqueous portion was 

transferred to 50-ml tube. The DNA was precipitated with 1x CTAB. After 

precipitation the DNA were hooked and dissolved in high salt TE. Adding 95% 

ethanol and the DNA were transferred to 1.5-ml microfuge tubes made final 

precipitation. The DNA was washed with 70% ethanol and was air-dried. After 

complete drying, two hundred µl of TE was added to dissolve the DNA. One µl of the 

DNA was loaded in 1% agarose gel along with 100 ng, 300 ng, 500 ng and 1000 ng 

concentration markers were eletrophoresed in 0.5x TBE buffer to determine the 

concentration of the DNA (Rogers and Bendich, 1994). 

4. Genes specific primer design and amplification 

The anthocynin specific primer pairs will be genereted from the DNA 

sequence of the anthocyanin structural genes (dihydroxyfravonol –4 reductase (DFR), 

anthocyanidin synthase (ANS), and the anthocyanin regulatory genes (Booster1 (B1; 

OsB1)) which involved anthocyanin biosynthesis in Oryza sativa L. (Gene Bank 

accession number AB003495, Y07955 and AB021079). All primer were designed by 

using Primer 3 Test Pre-Release output on a webservice, http://www- 

genome.wi.mit.edu/cgi-bin/primer /primer3_www_results.cgi, with the following 

parameters 40-60 %G+C rich, 60°C annealing temperature and 21 base pair in length. 

PCR reaction mixture volumes were 25 µL, containing 2.5 µl of 10x buffer, 25mM 

MgCl2, 200µM each of dNTPs (promega), 0.2µM anthocyanin specific primer, 100 

ng genomic DNA and 1 U Taq polymerase (promega). PCR amplification was 

performed in a Perkin Elmer Cetus Gene Amp PCR system 9700. The PCR products 

were electrophoresed on 1% agarose gels with 1x TBE and stained with ethidium 

bromide to verify amplification. 

16

5. Single-strand conformational polymorphism (SSCP) 

Five µL of the PCR product was mixed with 5 µl of denaturing solution (980 

ml/lite formamide, 50 ml/lite 0.2 M NaOH, 0.5 g/lite bromphenol blue, 0.5 g/lite 

xylene cyanol), heated for 5 min at 95°C, and quenched on ice. The samples were 

loaded onto the 8 % polyacrylamide/bis-acrylamide 99:1 and 1xTBE buffer. 1x TBE 

buffer (89 mM Tris-borate and 2 mM EDTA pH 8.0) was used as running buffer. 

Electrophoresis was performed at constant power, 5 watt, at 25°C for 18 hours. 

Polyacrylamide gels were silver-stained. 

6. RNA Analyses 

For expression analyses in developing grain of rice sample, during 10 day 

after pollination. Tissue samples were ground in liquid nitrogen, and total RNA was 

extracted with the high pure RNA isolation kit (Roche, Germany) according to the 

instructions of the manufacturer. The extracts were treated with 30 units of RNasefree 

DNase I (Roche, Germany). Two hundred ng of DNA-free RNA extract was 

converted into first-strand cDNA by using the One-step RT-RCR system (Roche, 

Germany) and 0.2 µM of each gene-specific primer. RT-PCR products were sizeseparated 

on a 1% (w/v) agarose gel. 

7. Analysis nucleotide sequencing of anthocyanin biosynthetic genes 

Anthocyanin biosynthetic genes PCR products will be used as templates for 

sequencing with big dye terminator sequencing kit (Perkin Elmer) and the ABI377 

automated DNA sequencer (Perkin Elmer). All sequencing reactions will be 

performed by DNA Fingerprinting Unit of Kasetsart University Khamphaengsaen. 

Sequencing for each sample will be carried out in both forward and reverse directions 

using anthocyanin biosynthetic genes specific primer forward and reverse sequencing 

primer respectively. 

17

8. Place and Duration 

This research was conducted at the Center of Excellence for Rice Molecular 

Breeding and Product Development, National Center for Agricultural Biotechnology, 

Kasetsart University, Kamphangsaen, Nakorn Pathom, Thailand from May 2001 to 

May 2005. 

18

Isolation of seed color mutants 

RESULTS 

Anthocyanin is a major class of flavonoids that accumulates in a variety of 

plant tissue in response to developmental and environmental signals. Mutant with 

novel expression pattern are readily identifiable, and have long been a subject of 

genetic studies. Characterization of those mutants has led to identification of genes 

encoding not only the enzyme of the anthocyanin biosynthetic pathway, but also the 

regulatory element that confer tissue-specific accumulation of anthocyanin (Holton 

and Cornish, 1995). The original rice, Jao Hom Nin rice (JHN) is nonglutiose that has 

purple pericarp, but mutant of JHN (BW1-4) have varying quantifying total 

anthocyanin in pericarp (Figure 3). How ever, there is white and varying purple 

pericarp color intensity in which mutants can led to identification of regulatory gene 

contronlling grain anthocyanin pigmentation in rice. 

Figure 3 Mutation of JHN seed color (BW1-4). 

To study BW (1-4) are mutation from JHN. DNA from 7 varity of rice 

(KDML105, KDxJHN, JHN, DH-JHN, Hei bao, BW1 and BW4) send to DNA 

19

Technology Laboratory use template for rice fingerprint by mulipex SSLP fluorcencelabel 

method on 12 chromosome number 78 locus. The multiplex PCR use 2-6 primer 

amplify DNA fragment and to examine size by program GeneScan (Applied 

Biosystem). Data show in appendix. Select 34 marker which have polymorphism 

allele score 1 and 0 to constructed a rice phylogentic tree by NTSYSpc 2.10 (Figure 

4). 

Figure 4 Phylogenetic tree of seven rice varieties using the NTSYSpc 2.10. 

From 34 polymorphism markers, phylogenetic tree of seven rice varieties 

(JHN, JHN(DH), Hei bao, BW1, BW4, KDML105 and an KDxJHN offspring) was 

constructed by NTSYS program. For the result, The phylogenetic tree of seven rice 

varieties separated into four groups at coefficient higher than 0.74. JHN group was 

consisted by JHN, double haploid of JHN, and Hei bao, sort of JHN. The nearest 

group is BW consisted by BW1 and BW1, mutants of JHN. Other two groups are the 

offspring of KDxJHN at coefficient 0.53 and KNML105 at coefficient 0.12, 

respectively. The result suggest that BW1 and BW4 are mutants that mutated from 

20

JHN since BW1 and BW4 had the fringerprinting result close JHN at coefficient 0.74 

more than the offspring of JHN and KDML105 that is coefficients 0.53 but less than 

Hei bao that is sort of JHN. 

Quantitative of Anothocyanin 

A simple, rapid method for determining total anthocyanins was developed for 

use in developing rice cultivars with dark-purple grains. The method was evaluated 

as absorbance was read at 535 nm and calculated as C = A x 288.21 (data show in 

method) where C is concentration of total anthocyanin (mg/kg), A is absorbance 

reading. Data show in table 2 and varying extractability of rice anthocyanin 

pingmentation see in Figure 5. The process was ued to determine total anthocyanins in 

pigmented rice. The maximum of total anthocyanins averaged 562.79 mg/kg in JHN 

(winter) but in summer has decrease total anthocyanins 112.98 mg/kg to indicate that 

temperature sensitive anthocyanin accumulation. White seed rice (KDML and JHN) 

has little synthesis anthocyanin. 

21

Table 2 Anthocyanin content of the rice varieties. 

Rice Sample Anthocyanins 

(mg/kg) 

KDML105 1.44 

JHN (summer) 112.98 

JHN (winter) 562.79 

BW1 0.48 

BW2 11.62 

BW3 3.94 

BW4 8.74 

JHN#3 (summer) 455.18 

DH 197.81 

KDML105 JHN(S) JHN(W) BW1 BW2 BW3 BW4 JHN#3 DH 

Figure 5 Varying extractability of rice anthocyanin pigmentation. 

Anthocyanin accumulation during grain development 

After fertilization, the grain anthocyanin levels were increased linearly to 10 

days before maturity in purple rice. When the grain anthocyanin content were 

determined at 5, 10, 15, 20, 25 and 30 days after fertilization, the effects of 

temperature during the grain filling period become clear (Figure 6). In winter, the 

grain anthocyanin accumulated much faster than in summer (562.79 mg/kg vs 112.98 

mg/kg, respectively) and maximized at 20 days after pollination. After 20 days, the 

accumulation of anthocyanin contents in both winter and summer were diminished at 

the similar rate. This later step in anthocyanin accumulation may be regulated by the 

22

increased degradation while the synthesis were down-regulated. The apparent 

anthocyanin intensities observed in the field 10 days before maturity were critical to 

the final grain anthocyanin content. 

Absorbance at 535 nm 

2.5 

2 

1.5 

1 

0.5 

0 

Comparison between anthocyanin with proanthocyanidin 

accumulation during rice grain development 

5 10 15 20 25 30 

Developmental stage 

5 10 15 20 25 30 

Figure 6 Anthocyanin accumulation during grain development in JHN purple rice. 

The developmental stages determined according to days after fertilizationstage 

6 day, 10 day, 15 day, 20 day, 25 day, 30 day. 

Regulatory Anthocyanin and Proanthocyanidin genes 

Defining region of rice seed color 

A (summer) 

PA (summer) 

A (winter) 

PA (winter) 

winter 

summer 

Seed color locus has been previously mapped on chromosome 4 from 

23

genetic map based on the 188 individuals from KDML105 and JHN was constructed 

using 114 SSR markers. The total map distance is 1,383.3 cM with an average 

interval distance of 12.1 cM. Grain color was scored in the F3 seeds using 1-3 scales. 

Two closely linked QTLs for seed color were detected on The two QTLs located 

within RM317-RM241 and RM252-RM241 marker intervals were accounted 

for58.3% and 56.7% of phenotypically variance explained (PVE), respectively. JHN 

alleles confer dark pericarb color on both QTLs. Multiloci QTL accounted for 64.5% 

of PVE (personal communicated). Computational gene finding programs found 

RM241 contained in OSJNBa0011L07 BAC distance 43 BAC contigs with RM317 

contained in OSJNBa0011J08 BAC and distance 8 BAC contigs with 

OSJNBa0065o17 that which contained a predictable OSB1 and OSB2 genes 

sequences (Figure 7). 

Figure 7 Location of gene controlling rice grain color. 

24

Genomic organization of OSB1 and OSB2 

To examine the OSJNBa0065o17 BAC of OSB1 and OSB2. Sequencing of 

this BAC revealed that the OSB1 coding region spanned approximately 6.3 kb, 

consisting of 11 exon and 10 introns. In addition to these regions, the gemomic 

organization of OSB1 was similar to that of Ra1, base on exon/intron boundaries and 

intron sequence such as the repeated sequence (Hu et al., 1996). In this BAC, the 

genomic organization of OSB2 was revealed to span approximately 24 kb with eight 

exon. It is notable the introns 2 and 6 of OSB2 are extraordinarily large (6.6 kb and 

~14 kb, respectively). Structure of OSB1 and OSB2 was demonstrated in Figure 8. 

2-bp deletion in OSB1 generated a frame shift 

Sequence of OSB1 and OSB2 cDNAs showed overall similarity with the maize 

R gene. The 2.2-kb OSB1 cDNA containd a 588- acid open reading frame (ORF), and 

showed almost complete identify (99.2%) to rice Ra1, and R-like gene previously 

reported by Hu et al., 1996. However, a 2-bp deletion in OSB1 generate a frame shift, 

affecting the 44 amino acids at it C-terminus relative to Ra1. In addition to the 

deletion, there were 11 single-nucleotide differences between OSB1 and Ra1, which 

resulted in seven amino acid substitutions upstream of the frame shift (Model 

structure shown in Figure 8) 

Design OBS1-A primer cover 2-bp deletion using select rice grain 

anthocyanin and proanthocyanidin biosynthesis trait. The amplified OSB1-A 

fragment on SSCP indentical which accumulated anthocyanin and proanthocyanidin 

in seed (SSCP of amplified OSB-1 fragment in Figure 10 and Sequencing of OSB-1 

fragment in Figure 9). 

25

Figure 8 The structure of OSB1 and OSB2 genes. Sequencing of OSB1 coding region 

spanned approximately 6.3 kb, consisting of 11 exon and 10 introns. In 

additional exon 11 have 2-bp deletion to generated a frame shift. 

Sequencing of OSB2 was revealed to span approximately 24 kb with eight 

exon and 7 introns. The introns 2 and 6 of OSB2 are extraordinarily large 

(6.6 kb and ~14 kb, respectively). 

26

JHN TAGCCTACCTCAAAGAGCTGGAGAAAAGAGTGGAAGAGCTGGAATCCAGCAGCCAACCAT 359 

Nipponbare TAGCCTACCTCAAAGAGCTGGAGAAAAGAGTGGAAGAGCTGGAATCCAGCAGCCAACCAT 359 

Murasaki TAGCCTACCTCAAAGAGCTGGAGAAAAGAGTGGAAGAGCTGGAATCCAGCAGCCAACCAT 92 

KDML105 TAGCCTACCTCAAAGAGCTGGAGAAAAGAGTGGAAGAGCTGGAATCCAGCAGCCAACCAT 360 

************************************************************ 

JHN CGCCATGTCCATTGGAAACAAGAAGCAGGCGAAAGTGCCGTGAGATCACTGGGAAGAAGG 419 

Nipponbare CGCCATGTCCATTGGAAACAAGAAGCAGGCGAAAGTGCCGTGAGATCACTGGGAAGAAGG 419 

Murasaki CGCCATGTCCATTGGAAACAAGAAGCAGGCGAAAGTGCCGTGAGATCACTGGGAAGAAGG 152 

KDML105 CGCCATGTCCATTGGAAACAAGAAGCAGGCGAAAGTGCCGTGAGATCACTGGGAAGAAGG 420 

************************************************************ 

JHN TTTCTGCAGGAGCGAAGAGAAAGGCGCCGGCGCCGGAGGTGGCCAGCGACGACGACACCG 479 

Nipponbare TTTCTGCAGGAGCGAAGAGAAAGGCGCCGGCGCCGGAGGTGGCCAGCGACGACGACACCG 479 

Murasaki TTTCTGCAGGAGCGAAGAGAAAGGCGCCGGCGCCGGAGGTGGCCAGCGACGACGACACCG 212 

KDML105 TTTCTGCAGGAGCGAAGAGAAAGGCGCCGGCGCCGGAGGTGGCCAGCGACGACGACACCG 480 

************************************************************ 

JHN ACGGGGAGCGGCGCCATTGTGTGAGCAACGTGAACGTCACCATCATGGACAACAAGGAGG 539 

Nipponbare ACGGGGAGCGGCGCCATTGTGTGAGCAACGTGAACGTCACCATCATGGACAACAAGGAGG 539 

Murasaki ACGGGGAGCGGCGCCATTGTGTGAGCAACGTGAACGTCACCATCATGGACAACAAGGAGG 272 

KDML105 ACGGGGAGCGGCGCCATTGTGTGAGCAACGTGAACGTCACCATCATGGACAACAAGGAGG 540 

************************************************************ 

JHN TTCTCCTCGAGCTGCAATGCCAGTGGAAGGAATTGCTGATGACGAGAGTGTTCGACGCGA 599 

Nipponbare TTCTCCTCGAGCTGCAATGCCAGTGGAAGGAATTGCTGATGACGAGAGTGTTCGACGCGA 599 

Murasaki TTCTCCTCGAGCTGCAATGCCAGTGGAAGGAATTGCTGATGACGAGAGTGTTCGACGCGA 332 

KDML105 TTCTCCTCGAGCTGCAATGCCAGTGGAAGGAATTGCTGATGACGAGAGTGTTCGACGCGA 600 

************************************************************ 

2-bp deletion 

JHN TCAAGGGAGTCTCCCTGGA--TCCTCTCGGTGCAGGCATCAACATCGGATGGTCTCCTTG 657 

Nipponbare TCAAGGGAGTCTCCCTGGATGTCCTCTCGGTGCAGGCATCAACATCGGATGGTCTCCTTG 659 

Murasaki TCAAGGGAGTCTCCCTGGA--TCCTCTCGGTGCAGGCATCAACATCGGATGGTCTCCTTG 390 

KDML105 TCAAGGGAGTCTCCCTGGATGTCCTCTCGGTGCAGGCATCAACATCGGATGGTCTCCTTG 660 

******************* *************************************** 

JHN GACTGAAGATACAAGCCAAGGTCGTCATCTCAGCGGCTAAGTAGAGCTCGCAGCAGAAAT 717 

Nipponbare GACTGAAGATACAAGCCAAGGTCGTCATCTCAGCGGCTAAGTAGAGCTCGCAGCAGAAAT 719 

Murasaki GACTGAAGATACAAGCCAAG---------------------------------------- 410 

KDML105 GACTGAAGATACAAGCCAAGGTCGTCATCTCAGCGGCTAAGTAGAGCTCGCAGCAGAAAT 720 

******************** 

Figure 9 Sequencing of OSB1-A fragment contained 2-bp deletion. 

27

JHN 

Pl w / Pl w 

KDML105 

+/+ 

#3 

Pl w / Pl w 

BW1 

+/+ 

BW2 

Pl w / Pl w 

BW3 

Pl w / Pl w 

BW4 

Pl w / Pl w 

1 

+/+ 

Figure 10 Amplifed product OSB1-A fragment run SSCP on 8% acylamide gel. 

SSCP of Amplified OSB1-A fragment in seven rice varieties and nine F2 of 

KDML105xJHN using screen Pl w genotype which Pl w /Pl w allele related accumulation 

of anthocyanin and proanthocyanidin in pericarp of rice grain. +/+ allele related not 

storage anthocyanin and proanthocyanin in rice pericarp. 

Temperature-sensitive DFR transcript 

Genomic organization of DFR 

2 

+/+ 

To examine of the DFR gene, genes specific primer deigned for polymerase 

chain reaction from Gene Bank accession number AB003495 that generate DFR 

fragment from DNA of KDML105, JHN, #3 and BW1. Sequencing of amplified DFR 

fragment that coding region spanned approximately 2 kb, consisting of 3 exon and 2 

3 

Pl w /+ 

F2 (KDML105xJNH) 

3 

Pl w / Pl w 

4 

Pl w /+ 

5 

Pl w / Pl w 

6 

+/+ 

7 

Pl w / Pl w 

8 

Pl w /+ 

9 

Pl w / Pl w 

Genotype 

28

intron (Figure 11 ). The sequencing of amplified DFR fragment was assembly by 

RICE GENE THRESHER program and multiple sequence alignment by ClustalW 

(EMBL-EBI database) that showed in Appendix. 

Figure11 Structure of rice dihydroxyflavanol-4 reductase (DFR) gene including 

primer positions. 

DFR-1I 

Temperature directing transcription profiles of DFR 

To examine the effect of the temperature on DFR at transcription level, RT- 

PCR analysis were performed on total RNA isolated from four rice strains in both 

winter, summer, and/or rainy seasons. Three transcription profiles were detected from 

these treatments. In such white rice as KDML105 and white BW1, only single pattern 

was detected in both winter and summer (Figure 12). In purple JHN and purple BW4 

rice strains, two patterns were identified in summer, winter, and/or rainy seasons. The 

394 bp was the expected if the intron 1 was completely spliced (Figure 12). All 

purple rice strains contained the expected transcript in all seasons. On the other hand, 

506 bp transcripts but not 394 bp were identified in the white rice grains. Using the 

same primers, PCR on the genomic DNA of JHN revealed only 506 bp genomic 

fragment. Therefore, the 506 bp identified in all white rice strains were the results of 

unspliced transcripts whereas the 394 bp fragments in all purple rice grains from 

different seasons were the spliced transcripts of DFR. The DFR may not be 

functional in white rice. Moreover, in purple rice strains, both 506 and 394 bp 

fragments were detected in summer while the 506 bp was detected but much less 

intensified in rainy season in JHN (Figure 12). In this case, the unspliced transcripts 

29

appeared in JHN and the purple mutant in summer corresponded well with the lower 

anthocyanin contents in summer in these two rice strains. Therefore, high 

temperature may affect grain anthocyanin content by interfering with intron 1 splicing 

of DFR. 

Figure 12 Expression of DFR determined by RT-PCR revealed transcription profiles 

in summer and winter. 

Post-transcriptional regulation of DFR is still a black box 

To understand the effect of unspliced transcripts at the translational level, in 

silico translation of the cloned and predicted coding sequence of the white and purple 

rice strains were compared. In DFR y , the full-length coding sequence from Murasaki 

(accession AB003495) and the predicted coding sequence from KDML105 revealed a 

peptide of 303 a.a. In DFR x , the full-length coding sequence from Purpleputtu 

(accession Y07959) and the predicted coding sequence from JHN revealed the peptide 

of 373 a.a. The DFR y and DFR x were very similar except 40-78 a.a. insertion from 

30

intron 1 in DFR y (Figure 13). This Intron 1 is translatable and in-frame with the 

leading exon. However, the inclusion of intron 1 in the peptide created a premature 

stop codon at position +153 nt while in DFR x , the full-length peptide was not 

interrupted. The premature stop codons in the specially exon may cause nonsensemediated 

decay (Issiki et al., 2001) ;and subsequently no template was available for 

translation. In this case, no leucoanthocyanin available for the anthocyanin synthase 

and the grains become white. To identified the conserved motif responsible for 

ineffective splicing, genomic sequences were compared of this gene on the first intron 

splice sites. No single nucleotide variation was detected at splice sites. The results 

described here demonstrate that the ability to splice out the intron 1 is a temperaturedependent 

process. This temperature sensitivity is not involved mutation at their 

splice sites. One possibility is that the temperature-dependent phenotype of DFR x is 

affected by the stability of RNA-RNA or RNA-Proteins interaction (Sablowski and 

Meyerowitz, 1998; Berget, 1995). The role of transacting regulators such as C1, 

OsB1, and OsB2 on intron 1 splicing must be illucidated. In case of unspliced DFR y , 

C1, OsB1, and OsB2 were not expressed whereas in DFR x , these regulatory proteins 

were expressed. We postulate that the ability to splice intron 1 depend on the 

interaction between the regulatory proteins and snRNA in spliceosome. When a white 

rice was transform with the regulatory genes (C1, OsB1, and OsB2), the transgenic 

became purple grains (Sakamoto et al., 2001). 

31

Figure 13 Two alleles of DFR, DFR x and DFR y .DFR x is temperature-sensitive allele 

that found in deeppurple grain (a, b). DFR y is the unspliced allele found in 

normal white rice (c). 

ANS, a key reaction for coloring in anthocyanin biosynthesis 

The reaction leading from colorless leucoanthocyanidin to anthocyanidin and 

its 3-O-glucoside is the critical step in the formation of colored metabolites in 

anthocyanin biosynthesis. In this study, ANS gene specific primer designed for 

polymerase chain reaction from Gene Bank accession number Y07955. DNA from 

KDML105, JHN, BW1, BW2, BW3, and BW4 were used as source of ANS for gene 

amplification (Figure 14). SSCP was used to screen ANS PCR-products for mutation 

in genomic sequence. The amplified ANS3 fragment will be used to determine the 

sequence variation related with anthocyanin pigmentation in giving various pericarp 

color (Figure 15) and Sequencing of ANS3 fragment shown in Figure 16. 

32

Figure 14 Structure of rice anthocyanidin synthase (ANS) gene including primer 

positions. 

KDML105 

JHN 

BW1 

Figure 15 PCR-SSCP method detecting anthocyanin biosynthetic genes mutation. 

BW2 

ANS3 fragment shown several pattern of SSCP in six rice cultivars. 

BW3 

BW4 

33

JHN CGCAAAGTTGTTCAAGAAGCTCAAGGATCAGCAAAACAACAATGCCGCAGCTGCATCGAA 60 

BW2 CGCAAAGTTGTTCAAGAAGCTCAAGGATCAGCAAGACAACAATGCCGCAGCTGCATCGAA 60 

KDML105 CCC-AAGCTGTTCAAGAAGCTCAAGGATCAGCAAGACAACAATGCCGCAGCTGCATCGAA 59 

BW1 CGCAAAGTTGTTCAAGAAGCTCAAGGATCAGCAAGACAACAATGCCGCAGGTGCATCGAA 60 

BW3 CGCAAAGTTGTTCAAGAAGCTCAAGGATCAGCAAGACAACAATGCCGCAGGTGCATCGAA 60 

BW4 CGCCAAGCTGTTCAAGAAGCTCAAGGATCAGCAAGACAACAATGCCGCAGGTGCATCGAA 60 

* * *** ************************** *************** ********* 

JHN CGGAATGATAACTAAATAATTGCAATTAGTCGATCTATCGGCGAGACAACCCAATATTTT 120 

BW2 CGGAATGATAACTAAATAATTGCAATTAGTCGATCTATCGGCGAGACAACCCAATATTTT 120 

KDML105 CGGAATGATAACTAAATAATTGCAATTAGTCGATCTATCGGCGAGACAACCCAATATTTT 119 

BW1 CGGAATGATAACTAAATAATTGCAATTAGTCGATCTATCGGCGAGACAACCTAATATTTT 120 



*************************************************** ******** 

JHN GAAAAATTGATGGACACACAAAAAAAAACTTTTTTATATAAGAATATGCATTTGGTTGAT 180 

BW2 GAAAAATTGATGGACACACAAAAAAAAACTTTTTTATATAAGAATATGCATTTGGTTGAT 180 

KDML105 GAAAAATTGATGGACACACAAAAAAAA-CTTTTTTATATAAGAATATGCATTTGGTTGAT 178 

BW1 GAAAAATTGATGGACACAAAAAAAA---CTTTTTTATATAAGAATATGCATTTAGTTGAT 177 



****************** ****** ************************* ****** 

JHN TCAACATGAAAAA-TATTTTCAAACCATTATATTTTTAATTGGTTATGAATAAACTATAA 239 

BW2 TCAACATGAAAAA-TATTTTCAAACCATTATATTTTTAATTGGTTATGAATAAACTATAA 239 

KDML105 TCAACATGAAAAA-TATTTTCAAACCATTATATTTTTAATTGGTTATGAATAAACTATAA 237 

BW1 TCAACATGAAAAAATATTTTCAAACCATTATATTTTTAATTTGTTATGAATAAACTATAA 237 



************* *************************** ****************** 

JHN AAATAATTGCATTCGTCTGAATTTCAGATAAAGGAGAACAAATAATGTTCAACGGAAGCG 299 

BW2 AAATAATTGCATTCGTCTGAATTTCAGATAAAGGAGAACAAATAATGTTCAACGGAAGCG 299 

KDML105 AAATAATTGCATTCGTCTGAATTTCAGATAAAGGAGAACAAATAATGTTCAACGGAAGCG 297 

BW1 AAATAATTGCATTCGTCTGAATTTTAGATAAAGGAGAACAAATAATGTTCATCAGAAACG 297 



************************ ************************** * *** ** 

JHN AAAGCCGGGGAAATGTACGGATGTTATATCGAGAGCTTAACTTATGAGATGTCTTATATT 359 

BW2 AAAGCCGGGGAAATGTACGGATGTTATATCGAGAGCTTAACTTATGAGATGTCTTATATT 359 

KDML105 AAAGCCGGGGAAATGTACGGATGTTATATCGAGAGCTTAACTTATGAGATGTCTTATATT 357 

BW1 AAAGCCAGGGAAACGTACGGATGTTATATCAAGAGTTTAACTTATGAGATGTCTTATATT 357 



****** ****** **************** **** ************************ 

JHN TTGTTGTCTATGTATTGCAGTCGACTTGTACACCGA 395 

BW2 TTGGTGTCTATGTATTGCAGTCGACTTGTACACCGA 395 

KDML105 TTGTTGTCTATGTATTGCAGTCGACTTGTACACCGA 393 

BW1 TTGTTGTCTATGTATTGCAGTCGACTTGTGCACCGA 393 

BW3 TTGTTGTCTATGTATTGCAGTCGACTTGTGCACCGA 393 

BW4 TTGTGGTCTATGTATTGCAGTCGACTTGTGCACCGA 393 

*** ************************ ****** 

Figure 16 DNA sequence alignment of ANS3 from different rice strain. The 

cladogram of amplified ANS3 fragment will be used to determine the 

sequence variation related with anthocyanin pigmentation in giving 

various pericarp color. To divide three group which group one is high 

antocyanin accumulation, group two and three are low accumulated 

anthocyanin (Figure 17). 

34

Figure17 Cladogram sequencing of ANS3 fragment. 

35

DISCUSSION 

OSB1 gene controlling grain anthocyanin and proanthocyanidin content in rice 

In the present study we characterized two rice genes, OSB1 and OSB2, with 

extensive homology to product of the maize R gene family which regulate 

anthocyanin and proanthocyanidin pigmentation. Several lines of evidence suggest 

that OSB1 comprise the Pl w allele. Based on these observations, we conclude that 

OSB1 is major components of the Plw allele. Our assumption that rice Pl might be 

orthologous to maize B enabled us, using B as a heterogous probe, to identify OSB1 

and OSB2. The notion that Pl is orthologous to B comes form the systeny between 

chromosome 2 in maize and chromosome 4 rice, where some morphological marker 

are also share (Ahn and Tanksley, 1993). B and R are displaced on two syntenic 

chromosome regions of maize (chromosome 2 and 10, respectively) that been 

evolutionarily duplicated by a polyploidization event. Characterization of the Pl w 

allele in this study that its complex organization rather resembles some alleles of the R 

locus. For example, the R-r allele contains three genes, P, Sl, and S2, by which plant 

and seed-specific expression is controlled, respectively (Robins et al., 1991). Further 

characterization of other Pl alleles is necessary to understand the diverse organization 

and differential expression of gene components. 

Anthocyanin intensity in rice grain is regulated by splicing efficiency of 

dihydroflavonol-4-reductase (DFR) is temperature sensitive 

To understand the genetic mechanism regulating splicing in DFR, genomic 

sequences around the exon1-2 junction from DFR x and DFR y were compared. 

However, genomic sequence around the splice junction of the two alleles were 

identical. Analysis of domain structure in the C1 using NCBI conserved domain 

found REB1, the mRNA splicing factor domain, located between a.a. 13-112 in the 

C1 of purplepittu rice (www.ncbi.nlm.nih.gov/structure/cdd/). This finding bring out 

one possibility that C1 may function as splicing factor specifically for the DFR 

36

(Figure 18). This hypothesis was supported by Saitoh et al. (2001). In this study the 

stable transgenic white rice (Tp309) with C1 gene from color maize produced purple 

seeds. However, the expression of DFR in the transgenic plants was not monitored. 

In arabidopsis, the temperature-sensitive mutant for floral homeotic genes, ap3-1, was 

caused by unstable base pairing between mRNA and snRNAs (Sablowski and 

Meyerowitz, 1998). The temperature-sensitivity of the anthocyanin accumulation 

open a unique opportunity to study the regulation of both transcription and posttranscription 

by a single regulatory protein. Understanding of this defective 

processing may point out ways to develop a more intensive anthocyanin content in 

rice grains without less in summer where high temperature is not permissive to 

produce anthocyanin-densed grains. 

Chromosome 6 

Figure 18 Comparison between REB1 domain of C1 Nipponbare with C1 Purpleputu. 

REB1, Myb superfamily proteins, including transcription factors and 

mRNA splicing factors (Transcription /RNA processing and modification / 

Cell division and chromosome partitioning) and temperature sensitive. 

37

ANS3 fragment determine the sequence variation related with anthocyanin 

pigmentation 

The amplified ANS3 fragment will be used to determine the sequence 

variation related with anthocyanin pigmentation in giving various pericarp color. 

Anthocyanidin synthase (ANS) is a non-haem iron(II)-dependent dioxygenase 

reported to catalyse the conversion of leucoanthocyanidins to anthocyanidins (Figure 

19) (Nakajima et al., 2001). Anthocyanidins are precursors of anthocyanins, which 

are a major family of pigments in higher plants. Mutation in this region would effect 

anthocyanin biosynthesis. Therefore, detection of variation in ANS gene may be used 

for identification of low anthocyanin plants. The structure of ANS provides a template 

for the ubiquitous family of plant nonhaem oxygenases for future engineering and 

inhibition studies. 

38

Figure 19 Mechanism of anthocyanin formation, leucoanthocyanidin to 

anthocyanidin 3-glucoside, catalyzed by ANS and 3-GT, and transport to 

vacuoleds (Nakajima et al., 2001). 

39

CONCLUSION 

Anthocyanin and proanthocyanidin biosyntheses in rice are regulated by either 

regulatory or structural genes. A regulatory gene, OSB1, which was in silico mapped 

near the major QTL for rice grain pericarp color on chromosome 4, was investigated 

between KDML105 (white rice) and Jao Hom Nin (JHN, deep-purple rice). Sequence 

analysis revealed that 2-bp deletion found in the exon 11 of this gene in JHN was 

associated with the pericarp color. DFR, an important structural gene, is necessary for 

anthocyanin biosynthesis. The expression of this gene was highly regulated by 

temperature. In JHN, DFR highly expressed at low temperature (20 °C) but was 

suppressed at high temperature (34 °C). Moreover, 506-bp pre-mRNA containing the 

intron 1 was detected at high temperature in JHN and it mutant with brown pericarp, 

BW4. On the other hand, in KDML105 and BW1, a JHN mutant with white pericarp, 

506-bp mature mRNA was not detected; only pre-mRNA containing the intron 1 was 

accumulated. Another structural gene, ANS, is a key gene reacting for coloring in 

anthocyanin biosynthesis. Sequence analysis of the ANS3 fragments, 3’ UTR of this 

gene, among rice strains that have pericarp colors varying from white to deep-purple 

revealed that these strains can be grouped according to the sequence variations found 

in this region. Understanding the genetic bases of anthocyanin and proanthocyanidin 

pigmentation is helpful for rice breeders to improve rice grain color as well as plant 

molecular biologists to develop a regulatory anthocyanin gene as a reporter in plant 

transformation. 

40

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45

APPENDIX 

46

Multiplex SSLP fluorescence-labeled 

RM 243 

RM212 

RM 246 

RM 259 

APPENDIX 

RM 104 

Graphical of SSLP marker on chromosome 1 deteced by program GeneScan 

(Aplied Biosystem) 

RM 110 

RM 29 

RM 341 

RM 250 RM 53 

RM146 RM 166 



47

RM 132 

RM 85 



RM 335 

RM 282 

RM 7 

RM 323 

RM 231 

RM 16 

RM 273 

RM 261 RM 252 RM 142 

RM 131 

RM 317 

RM 280 

RM 55 



48

RM 178 

RM 13 

RM 173 

RM 334 

RM169 

RM 164 



RM 111 

RM 00 

RM 253 

RM 121 

RM 162 

RM 204 

RM 103 



49

RM 214 

RM 2 

RM 125 RM 234 

RM 172 

RM 182 



RM 256 

RM 44 

RM 310 

EO3-92.0 

RM 337 RM 38 

RM 264 



50

aa 

RM 205 

RM 257 

RM 105 

RM 215 

RM 219 

RM 242 



RM 294 

RM 147 

RM 244 

RM 239RM 222 

RM 271 

RM 171 



51

RM 286 

RM 206 

RM 287 

RM 202 



RM 235 

RM 247 

RM 309 

RM20 

RM 332 

RM 20 

RM 139 



52

RM 132 RM 232 RM 7 



Blue = florescence-labeled primer with Fam 

Green = florescence-labeled primer with Tet 

Black = florescence-labeled primer with Hex 

RM 231 

53

SSLP marker fluorescence-labeled 

marker size rang (bp) fluorescencelabeled 

Chromosome 1 RM259 156-175 Hex 

RM5 108-130 Tet 

RM246 97-118 Hex 

RM212 112-134 Fam 

RM104 222-238 Fam 

Chromosome 2 RM110 138-156 Fam 

RM53 168-208 Fam 

RM29 188-200 Tet 

RM341 136-172 Tet 

RM106 288-297 Fam 

RM250 154-174 Hex 

RM166 203-217 Hex 

Chromosome 3 RM132 83-83 Tet 

RM231 169-191 Hex 

RM7 168-182 Fam 

RM232 142-166 Tet 

RM282 128-136 Fam 

RM16 168-230 Tet 

RM55 217-235 Fam 

RM85 85-107 Fam 

54

(Continued) 



RM261 122-126 Fam 

RM142 235-238 Fam 

RM273 199-207 Fam 

RM252 193-262 Tet 

RM317 146-166 Fam 

RM131 209-217 Hex 

RM280 148-181 Hex 


RM169 164-194 Hex 

RM164 246-304 Fam 

RM173 186-188 Tet 

RM178 117-123 Fam 

RM334 146-197 Fam 


RM204 106-194 Fam 

RM111 118-148 Tet 

RM253 217-239 Hex 

RM121 258-264 Tet 

RM162 119-189 Hex 

RM103 334-340 Fam 


RM214 114-152 Tet 

RM2 144-153 Hex 

RM182 336-346 Fam 

RM234 133-163 Hex 

RM172 159-165 Fam 

55

(Continued) 



RM38 246-278 Fam 

RM310 85-120 Fam 

RM44 93-131 Tet 

E03-92.0 200 Hex 

RM256 107-139 Hex 

RM264 148-178 Tet 


aa 

(126G1R) 

100-118 Fam 

RM105 131-140 Fam 

RM257 121-173 Tet 

RM242 193-225 Hex 

RM215 147-153 Fam 

RM205 123-161 Hex 


RM244 157-165 Fam 

RM239 164-186 Tet 

RM271 92-105 Fam 

RM171 318-343 Hex 

RM294 

(A,B) 

100-180 Hex 

RM147 94-97 Tet 

Chromosome 11 RM286 99-128 Hex 

RM332 162-183 Tet 

RM202 158-186 Hex 

RM287 98-118 Tet 

RM206 128-202 Fam 

RM139 396-410 Fam 

56

(Continued) 


Chromosome 12 RM20(A,B) 162-198, 116- 

140 

Fam 

RM247 130-176 Hex 

RM309 165-169 Tet 

RM235 96-134 Tet 

57

Primer used in overlapping strategy for entirely sequencing of the rice 

anthocyanin biosynthetic genes 

OSB1-AF 5’ GAGAAGCTCAACGAGATGTT 3’ 

OSB1-AR 5’ CTAGCTAGCTAGCTTGCTATAGC 3’ 

ANS1-F 5’ CACGCAGCTCATCTACTAGC 3’ 

ANS1-R 5’ AGTTGATCTTGAGCTGGAGG 3’ 

ANS2-F 5’ GCCTGAGAGGACATGAGCTG 3’ 

ANS2-R 5’ GTTATCATTCCGTTCGATGC 3’ 

ANS3-F 5’ GCAACGCAAGCTGTTCAAG 3’ 

ANS3-R 5’ GGGATGGAAAGACTCGGTG 3’ 

DFR1-F 5’ AAACCGGTTCATTCTGTCTC 3’ 

DFR1-R 5’ GCAAGCGGCATATATAATTG 3’ 

DFR2-F 5’ ATATATAGGCGAGCCAACG 3’ 

DFR2-R 5’ CGTTTCGTTTTATGTACGTG 3’ 

DFR3-F 5’ CGTTCAAACTCACCCTTAAG 3’ 

DFR3-R 5’ GTGAAATAGACGAAGCCAAC 3’ 

DFR4-F 5’ CGTTGGTCAAATGAGTGTTG 3’ 

DFR4-R 5’ CACGACTTAGAATCGCACAC 3’ 

DFR-1IF 5’ CTCGTCATGAAGCTCCTC 3’ 

DFR-1IR 5’ AGTCGATGTCGCTCCAGT 3’ 

58

CLUSTAL W (1.82) multiple sequence alignment of DFR gene 

KDML105_DNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

BW1_DNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

Mura_DNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

Nip_DNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

Mura_cDNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

Purple_cDNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

JHN_DNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

#3_DNA TGCGAATCCAACACAAGCACCGCGGCGTAGTACTACTACTTGCGCGCGCGTGTTAGATTC 60 

************************************************************ 

KDML105_DNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

BW1_DNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

Mura_DNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

Nip_DNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

Mura_cDNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

Purple_cDNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

JHN_DNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

#3_DNA GCGTGCGAATCCAACACAAGCAGATCGATCACGCACGGTACGCCATGGGCGAGGCGGTGA 120 

************************************************************ 

KDML105_DNA AGGGGCCAGTGGTGGTGACGGCCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

BW1_DNA AGGGGCCAGTGGTGGTGACGGGCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

Mura_DNA AGGGGCCAGTGGTGGTGACGGGCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

Nip_DNA AGGGGCCAGTGGTGGTGACGGGCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

Mura_cDNA AGGGGCCAGTGGTGGTGACGGGCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

Purple_cDNA AGGGGCCAGTGGTGGTGACGGGCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

JHN_DNA AGGGGCCAGTGGTGGTGACGGGCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

#3_DNA AGGGGCCAGTGGTGGTGACGGGCGCGTCGGGCTTCGTCGGCTCATGGCTCGTCATGAAGC 180 

********************* ************************************** 

KDML105_DNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCTGTGAGCTCTCTCATC 240 

BW1_DNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCTGTGAGCTCTCTCATC 240 

Mura_DNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCTGTGAGCTCTCTCATC 240 

Nip_DNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCTGTGAGCTCTCTCATC 240 

Mura_cDNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCT--------------- 225 

Purple_cDNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCT--------------- 225 

JHN_DNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCTGTGAGCTCTCTCATC 240 

#3_DNA TCCTCCAGGCCGGCTACACCGTCCGCGCCACAGTGCGCGACCCCTGTGAGCTCTCTCATC 240 

********************************************* 

KDML105_DNA GTGCACTCTAGCTCTCTCCTCGTAGTTTACTGACTCCAATTATATATGCCGCTTGCTTGA 300 

BW1_DNA GTGCACTCTAGCTCTCTCCTCGTAGTTTACTGACTCCAATTATATATGCCGCTTGCTTGA 300 

Mura_DNA GTGCACTCTAGCTCTCTCCTCGTAGTTTACTGACTCCAATTATATATGCCGCTTGCTTGA 300 

Nip_DNA GTGCACTCTAGCTCTCTCCTCGTAGTTTACTGACTCCAATTATATATGCCGCTTGCTTGA 300 

Mura_cDNA ------------------------------------------------------------ 

Purple_cDNA ------------------------------------------------------------ 

JHN_DNA GTGCACTCTAGCTCTCTCCTCGTAGTTTACTGACTCCAATTATATATGCCGCTTGCTTGA 300 

#3_DNA GTGCACTCTAGCTCTCTCCTCGTAGTTTACTGACTCCAATTATATATGCCGCTTGCTTGA 300 

KDML105_DNA CTCTGACAAGTGTACGTGTTGTTGTTGTTGTTTTCAGCTAACGTTGGGAAGACGAAGCCG 360 

BW1_DNA CTCTGACAAGTGTACGTGTTGTTGTTGTTGTTTTCAGCTAACGTTGGGAAGACGAAGCCG 360 

Mura_DNA CTCTGACAAGTGTACGTGTTGTTGTTGTTGTTTTCAGCTAACGTTGGGAAGACGAAGCCG 360 

Nip_DNA CTCTGACAAGTGTACGTGTTGTTGTTGTTGTTTTCAGCTAACGTTGGGAAGACGAAGCCG 360 

Mura_cDNA -------------------------------------CTAACGTTGGGAAGACGAAGCCG 248 

Purple_cDNA -------------------------------------CTAACGTTGGGAAGACGAAGCCG 248 

JHN_DNA CTCTGACAAGTGTACGTGTTGTTGTTGTTGTTTTCAGCTAACGTTGGGAAGACGAAGCCG 360 

#3_DNA CTCTGACAAGTGTACGTGTTGTTGTTGTTGTTTTCAGCTAACGTTGGGAAGACGAAGCCG 360 

*********************** 

59

KDML105_DNA TTGCTGGAGCTGGCGGGGTCGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 420 

BW1_DNA TTGCTGGAGCTGGCGGGGTCGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 420 

Mura_DNA TTGCTGGAGCTGGCGGGGTCGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 420 

Nip_DNA TTGCTGGAGCTGGCGGGGTAGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 420 

Mura_cDNA TTGCTGGAGCTGGCGGGGTCGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 308 

Purple_cDNA TTGCTGGAGCTGGCGGGGTCGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 308 

JHN_DNA TTGCTGGAGCTGGCGGGGTCGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 420 

#3_DNA TTGCTGGAGCTGGCGGGGTCGAAGGAGAGGCTGACGCTGTGGAAGGCCGACCTGGGCGAG 420 

******************* **************************************** 

KDML105_DNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 480 

BW1_DNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 480 

Mura_DNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 480 

Nip_DNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 480 

Mura_cDNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 368 

Purple_cDNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 368 

JHN_DNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 480 

#3_DNA GAAGGCAGCTTCGACGCGGCGATCAGGGGTTGCACGGGCGTGTTCCACGTCGCGACGCCC 480 

************************************************************ 

KDML105_DNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGATCAAGCCCACCGTGGAAGGGATG 540 

BW1_DNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGATCAAGCCCACCGTGGAAGGGATG 540 

Mura_DNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGGTCAAGCCCACCGTGGAAGGGATG 540 

Nip_DNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGGTCAAGCCCACCGTGGAAGGGATG 540 

Mura_cDNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGGTCAAGCCCACCGTGGAAGGGATG 428 

Purple_cDNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGATCAAGCCCACCGTGGAAGGGATG 428 

JHN_DNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGATCAAGCCCACCGTGGAAGGGATG 540 

#3_DNA ATGGACTTCGAGTCCGAGGACCCGGAGAACGAGGTGATCAAGCCCACCGTGGAAGGGATG 540 

************************************ *********************** 

KDML105_DNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 600 

BW1_DNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 600 

Mura_DNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 600 

Nip_DNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 600 

Mura_cDNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 488 

Purple_cDNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 488 

JHN_DNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 600 

#3_DNA CTGAGCATCATGCGGGCCTGCAGGGACGCCGGCACCGTCAAGCGCATCGTCTTCACCTCC 600 

************************************************************ 

KDML105_DNA TCCGCCGGGACCGTCAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCACGACGACTGG 660 

BW1_DNA TCCGCCGGGACCGACAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCACGACGACTGG 660 

Mura_DNA TCCGCCGGGACCGTCAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCACGACGACTGG 660 

Nip_DNA TCCGCCGGGACCGTCAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCACGACGACTGG 660 

Mura_cDNA TCCGCCGGGACCGTCAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCACGACGACTGG 548 

Purple_cDNA TCCGCCGGGACCGTCAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCACGACGACTGG 548 

JHN_DNA TCCGCCGGGACCGTCAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCTCGACGACTGG 660 

#3_DNA TCCGCCGGGACCGTCAACATCGAGGAGCGGCAGCGCCCCTCCTACGACCACGACGACTGG 660 

************* *********************************** ********** 

KDML105_DNA AGCGACATCGACTTCTGTCGCCGCGTCAAGATGACCCGATGGGTCTATATCGAGAATGTC 720 

BW1_DNA AGCGACATCGACTTCTGTCGCCGCGTCAAGATGACCCGATGGGTCTATATCGAGAATGTC 720 

Mura_DNA AGCGACATCGACTTCTGCCGCCGCGTCAAGATGACCGGATGGGTATGTATCGAAAATGTT 720 

Nip_DNA AGCGACATCGACTTCTGCCGCCGCGTCAAGATGACCGGATGGGTATGTATCGAAAATGTT 720 

Mura_cDNA AGCGACATCGACTTCTGCCGCCGCGTCAAGATGACCGGATGG------------------ 590 

Purple_cDNA AGCGACATCGACTTCTGTCGCCGCGTCAAGATGACCGGATGG------------------ 590 

JHN_DNA AGCGACATCGACTTCTGTCGCCGCGTCAAGATGACCCGATGGGTATGTATCGAAAATGTT 720 

#3_DNA AGCGACATCGACTTCTGTCGCCGCGTCAAGATGACCCGATGGGTATGTATCGAAAATGTT 720 

***************** ****************** ***** 

KDML105_DNA GTCCTGTGTTAAGAACCTCAATCCTTCACCTACATAATCGAAAACGATATCTTCCTCTGA 780 

BW1_DNA GTCCTGTGTTAAGAACCTCAATCCTTCACCTACATAATCGAAAACGATATCTTCCTCTGA 780 

Mura_DNA GTCGTGGGTTAGGAACAACGATCCTCCACGTACATAAAACGAAACGATAAGTTAACATGA 780 

Nip_DNA GTCGTGGGTTAGGAACAACGATCCTCCACGTACATAAAACGAAACGATAAGTTAACATGA 780 

Mura_cDNA ------------------------------------------------------------ 

Purple_cDNA ------------------------------------------------------------ 

JHN_DNA GTCGTGGGTTAGGAACAACGATCCTCCACGTACATAAAACGAAACGATAAGTTAACATGA 780 

#3_DNA GTCGTGGGTTAGGAACAACGATCCTCCACGTACATAAAACGAAACGATAAGTTAACATGA 780 

60

KDML105_DNA GCGTGATCAATATTAGTATGGTATAATTGATATTTGTTTAAAAATCTAAAAAATATTAAT 840 

BW1_DNA GCGTGATCAATATTAGTATGGTATAATTGATATTTGTTTAAAA-TCTAAAAAATATTAAT 839 

Mura_DNA GCATGATTAATATTAGTATGGTATAATTGATATTTGTTTAAAAATCTAAAAAATATTAAT 840 

Nip_DNA GCATGATTAATATTAGTATGGTATAATTGATATTTGTTTAAAAATCTAAAAAATATTAAT 840 

Mura_cDNA ------------------------------------------------------------ 

Purple_cDNA ------------------------------------------------------------ 

JHN_DNA GCATGATTAATATTAGTATGGTATAATTGATATTTGTTTAAAA-TCTAAAAAATATTAAT 839 

#3_DNA GCATGATTAATATTAGTATGGTATAATTGATATTTGTTTAAAA-TCTAAAAAATATTAAT 839 

KDML105_DNA ATGATTTTTAAATAACTATTTTATA-AATTTTTTTTATGAAAACACAAGGAAACAGAAAT 899 

BW1_DNA ATGATTTTTAAATAACTATTTTATAGAATTTTTTTTATGAAAACACAAGGAAACAGAAAT 899 

Mura_DNA ATGATTTTTAAATAACTATTTTATAGAATTTTTTTTATGAAAACACAAGGAAACAGAAAT 900 

Nip_DNA ATGATTTTTAAATAACTATTTTATAGAATTTTTTTTATGAAAACACAAGGAAACAGAAAT 900 

Mura_cDNA ------------------------------------------------------------ 

Purple_cDNA ------------------------------------------------------------ 

JHN_DNA ATGATTTTTAAATAACTATTTTATAGAATTTTTTTTATGAAAACACAAGGAAACAGAAAT 899 

#3_DNA GTGATTTTTAAATAACTATTTTATAGAATTTTTTTTATGAAAACACAAGGAAACAGAAAT 899 

KDML105_DNA TGAGAAATAGTACGTTCAAACTCACCCTTAAGCAACTGAAACTAGCTTAGCACGTGAATT 959 

BW1_DNA TGAGAAATAGTACGTTCAAACTCACCCTTAAGCAACTGAAACTAGCTTAGCACGTGAATT 959 

Mura_DNA TGAGAAATAGTACGTTCAAACTCACCCTTAAGCAACTGAAACTAGCTTAGCACGTGAATT 960 

Nip_DNA TGAGAAATAGTACGTTCAAACTCACCCTTAAGCAACTGAAACTAGCTTAGCACGTGAATT 960 

Mura_cDNA ------------------------------------------------------------ 

Purple_cDNA ------------------------------------------------------------ 

JHN_DNA TGAGAAATAGTACGTTCAAACTCACCCTTAAGCAACTGAAACTAGCTTAGCACGTGAATT 959 

#3_DNA TGAGAAATAGTACGTTCGAACTCACCCTTAAGCAACTGAAACTAGCTTAGCACGTGAATT 959 

KDML105_DNA TGGCCGTGCGAGTCATATGATATGAAGGTCGGGGAT-GTTTTTTTTTTTTGGGGGGGAGG 1018 

BW1_DNA TGGCCGTGCGAGTCATATGATATGAAGGTCGGGGACCGTTTTTTTTTTTTTGGGGGGATG 1019 

Mura_DNA TGGCCGTGTGAGTCATATGATATGAAGGTCGGGGATGTTTTTTTTTTTTTTGCGGGGATG 1020 

Nip_DNA TGGCCGTGTGAGTCATATGATATGAAGGTCGGGGATGTTTTTTTTTTTTTTGCGGGGATG 1020 

Mura_cDNA ------------------------------------------------------------ 

Purple_cDNA ------------------------------------------------------------ 

JHN_DNA TGGCCGTGCGAGTCATATGATATGAAGGTCGGGGAT-GTTTTTTTTTTTTGGGGGGGAGG 1018 

#3_DNA TGGCCGTGCGAGTCATATGATATGAAGGTCGGGGAT-GTTTTTTTTTTTTGGGGGGGAGG 1018 

KDML105_DNA TAATTAACTAATTTTGTAAACCATTTCTATTGTTTAAAAAAAGTTAGCAAGTGATAATGG 1078 

BW1_DNA TAATTAACTAATTTTGTAAACCATTTTTATTGGTTAAAAAAAGTTAGCAAGTGATAATTG 1079 

Mura_DNA TAATTAACTAATTATGTAAACCATTTCTATTGTCTAAAAGAAGTTAGCAAGTGATAATTG 1080 

Nip_DNA TAATTAACTAATTATGTAAACCATTTCTATTGTCTAAAAGAAGTTAGCAAGTGATAATTG 1080 

Mura_cDNA ------------------------------------------------------------ 

Purple_cDNA ------------------------------------------------------------ 

JHN_DNA TAATTAACTAATTATGGAAACCATTTTTATGGGTAAAAAAAAGTTAGCAGGGGGTAATTG 1078 

#3_DNA TAATTAACTAATTTTGTAAACCATTTCTATTGCTTAAAAAAAGTTACCAAGTTTTAATTG 1078 

KDML105_DNA GGGGGGCAGATGTACTTCGGGTCCAAGTCATTGGCCGAAAAGGCCGCCATGGAATACGCG 1138 

BW1_DNA GGGGGGCAGATGTACTTCGGGTCCAAGTCATTGGCGGAAAAGGCCCCCTTGGAATACGCG 1139 

Mura_DNA TGGTGGCAGATGTACTTCGTGTCCAAGTCATTGGCGGAGAAGGCCGCCATGGAATACGCG 1140 

Nip_DNA TGGTGGCAGATGTACTTCGTGTCCAAGTCATTGGCGGAGAAGGCCGCCATGGAATACGCG 1140 

Mura_cDNA ---------ATGTACTTCGTGTCCAAGTCATTGGCGGAGAAGGCCGCCATGGAATACGCG 641 

Purple_cDNA ---------ATGTACTTCGTGTCCAAGTCATTGGCGGAGAAGGCCGCCATGGAATACGCG 641 

JHN_DNA TGGGGGCAAATGTCCTTCGGGCCCAACCCATTGGGGGAAAAGGCCCCCTTGGAATACCCG 1138 

#3_DNA GGGGGGGGGATGTTCTTTGGGTCCAAGTAATTGGGGGAAAAGGCCCCCTTGGAATACCCG 1138 

**** *** * * **** ***** ** ****** ** ******** ** 

KDML105_DNA AGGGAGCACGGGCTGGACCTCATCAGCGTCATCCCCACGCTCGTCGTCGGGCCCTTCATC 1198 

BW1_DNA AGGGAGCACGGGTTGGACCTCATCAGCGTCATCCCCACGCTCGTCGTCGGGCCCTTCATC 1199 

Mura_DNA AGGGAGCACGGGCTGGACCTCATCAGCGTCATCCCCACGCTCGTCGTCGGGCCCTTCATC 1200 

Nip_DNA AGGGAGCACGGGCTGGACCTCATCAGCGTCATCCCCACGCTCGTCGTCGGGCCCTTCATC 1200 

Mura_cDNA AGGGAGCACGGGCTGGACCTCATCAGCGTCATCCCCACGCTCGTCGTCGGGCCCTTCATC 701 

Purple_cDNA AGGGAGCACGGGCTGGACCTCATCAGCGTCATCCCCACGCTCGTCGTCGGGCCCTTCATC 701 

JHN_DNA AGGGAGCCCGGGTTGGACCTTATAAGGGTAATCCCCACCCTCGTGGGGGGGCCTTTCATC 1198 

#3_DNA AGGGAGCCCGGGTTGGACCTTATAAGGGTAATCCCCACCCTCGTGGGGGGGCCCTTAATT 1198 

******* **** ******* ** ** ** ******** ***** * ***** ** ** 

61

KDML105_DNA AGCAACGGGATGCCGCCGAGCCACGTCACCGCGCTGGCGCTGCTCACGGGGAACGAGGCC 1258 

BW1_DNA AGCAACGGGATGCCGCCGAGCCACGTCACCGCGCTGGCGCTGCTCACGGGGAACGAGGCC 1259 

Mura_DNA AGCAACGGGATGCCGCCGAGCCACGTCACCGCGCTGGCGCTGCTCACGGGGAACGAGGCC 1260 

Nip_DNA AGCAACGGGATGCCGCCGAGCCACGTCACCGCGCTGGCGCTGCTCACGGGGAACGAGGCC 1260 

Mura_cDNA AGCAACGGGATGCCGCCGAGCCACGTCACCGCGCTGGCGCTGCTCACGGGGAACGAGGCC 761 

Purple_cDNA AGCAACGGGATGCCGCCGAGCCACGTCACCGCGCTGGCGCTGCTCACGGGGAACGAGGCC 761 

JHN_DNA AGCAACGGGATGCCGCCGAGCCACGTAACCGCGCTGGCGCTGCTCACGGGGAACGAGGCC 1258 

#3_DNA AGAAACGGGATGCCGCCGAGCCACGTAACCGGGCTGGGGTTGTTTACGGGGAACAAGGCC 1258 

** *********************** **** ***** * ** * ********* ***** 

KDML105_DNA CACTACTCGATCCTGAAGCAGGTGCAGTTCGTCCACCTCGACGACCTCTGCGATGCCGAG 1318 

BW1_DNA CACTACTCGATCCTGAAGCAGGTGCAGTTCGTCCACCTCGACGACCTCTGCGATGCCGAG 1319 

Mura_DNA CACTACTCGATCCTGAAGCAGGTGCAGTTCGTCCACCTCGACGACCTCTGCGACGCCGAG 1320 

Nip_DNA CACTACTCGATCCTGAAGCAGGTGCAGTTCGTCCACCTCGACGACCTCTGCGACGCCGAG 1320 

Mura_cDNA CACTACTCGATCCTGAAGCAGGTGCAGTTCGTCCACCTCGACGACCTCTGCGACGCCGAG 821 

Purple_cDNA CACTACTCGATCCTGAAGCAGGTGCAGTTCGTCCACCTCGACGACCTCTGCGATGCCGAG 821 

JHN_DNA CACTACTCGATCCTGAAGCAGGTGCAGTTCGTCCACCTCGACGACCTCTGCGATGCCGAA 1318 

#3_DNA CATTATTTGATCTTGAAGCAGGGGCAGTTGGTCCACCTCGACAACCTTTGGGATGCCGAA 1318 

** ** * **** ********* ****** ************ **** ** ** ***** 

KDML105_DNA ATCTTCCTCTTCGAGAGCCCCGAGGCGCGCGGCCGCTACGTCTGCTCCTCCCACGACGCC 1378 

BW1_DNA ATCTTCCTCTTCGAGAGCCCCGAGGCGCGCGGCCGCTACGTCTGCTCCTCCCACGACGCC 1379 

Mura_DNA ATCTTCCTCTTCGAGAGCCCCGAGGCGCGCGGCCGCTACGTCTGCTCCTCCCACGACGCC 1380 

Nip_DNA ATCTTCCTCTTCGAGAGCCCCGAGGCGCGCGGCCGCTACGTCTGCTCCTCCCACGACGCC 1380 

Mura_cDNA ATCTTCCTCTTCGAGAGCCCCGAGGCGCGCGGCCGCTACGTCTGCTCCTCCCACGACGCC 881 

Purple_cDNA ATCTTCCTCTTCGAGAGCCCCGAGGCGCGCGGCCGCTACGTCTGCTCCTCCCACGACGCC 881 

JHN_DNA ATTTTCCTCTTCGAGAGCCCCGAGGCGCGCGGCCGCTACGTCTGCTCCTCCCACGACGCC 1378 

#3_DNA ATTTTCTTTTTTAAAAGCCCCAAGGGGCGCGGCCGTTACGTTTGTTTTTCCCACAACGCC 1378 

** *** * ** * ****** *** ********* ***** ** * ****** ***** 

KDML105_DNA ACCATCCACGGCCTCGCGACGATGCTCGCGGACATGTTCCCGGAGTACGACGTGCCGCGG 1438 

BW1_DNA ACCATCCACGGCCTCGCGACGATGCTCGCGGACATGTTCCCGGAGTACGACGTGCCGCGG 1439 

Mura_DNA ACCATCCACGGCCTCGCGACGATGCTCGCGGACATGTTCCCGGAGTACGACGTGCCGCGG 1440 

Nip_DNA ACCATCCACGGCCTCGCGACGATGCTCGCGGACATGTTCCCGGAGTACGACGTGCCGCGG 1440 

Mura_cDNA ACCATCCACGGCCTCGCGACGATGCTCGCGGACATGTTCCCGGAGTACGACGTGCCGCGG 941 

Purple_cDNA ACCATCCACGGCCTCGCGACGATGCTCGCGGACATGTTCCCGGAGTACGACGTGCCGCGG 941 

JHN_DNA ACCATCCACGGCCTCGCGACGATGCTCGCGGACATGTTCCCGGAGTACGACGTGCCGCGG 1438 

#3_DNA ACCATTCACGGCCTTGGAACAATGTTTGGGGACATGTTCCCGGAGTACAACGTGCCGGGG 1438 

***** ******** * ** *** * * ******************* ******** ** 

KDML105_DNA AGCTTT-CCCGGGATCGACGCCGACCACCTCCAGCCGGTGCACTTCTCGTCGTGGAAGCT 1497 

BW1_DNA AGCTTT-CCCGGGATCGACGCCGACCACCTCCAGCCGGTGCACTTCTCGTCGTGGAAGCT 1498 

Mura_DNA AGCTTT-CCCGGGATCGACGCCGACCACCTCCAGCCGGTGCACTTCTCGTCGTGGAAGCT 1499 

Nip_DNA AGCTTT-CCCGGGATCGACGCCGACCACCTCCAGCCGGTGCACTTCTCGTCGTGGAAGCT 1499 

Mura_cDNA AGCTTT-CCCGGGATCGACGCCGACCACCTCCAGCCGGTGCACTTCTCGTCGTGGAAGCT 1000 

Purple_cDNA AGCTTT-CCCGGGATCGACGCCGACCACCTCCAGCCGGTGCACTTCTCGTCGTGGAAGCT 1000 

JHN_DNA AGCTTT-CCCGGGATCGACGCCGACCACCTCCAGCCGGTGCACTTCTCGTCGTGGAAGCT 1497 

#3_DNA AGCTTTTCCCGGGATCGACGCCGACCCCCTCCAGCCGGGGCATTTTTGGTGGGGGAAGCT 1498 

****** ******************* *********** *** ** * ** * ******* 

KDML105_DNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1557 

BW1_DNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1558 

Mura_DNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1559 

Nip_DNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1559 

Mura_cDNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1060 

Purple_cDNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1060 

JHN_DNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1557 

#3_DNA CCTCGCCCACGGGTTCAGGTTCAGGTACACGCTGGAGGACATGTTCGAGGCCGCCGTCCG 1558 

************************************************************ 

KDML105_DNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1617 

BW1_DNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1618 

Mura_DNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1619 

Nip_DNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1619 

Mura_cDNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1120 

Purple_cDNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1120 

JHN_DNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1617 

#3_DNA GACGTGCAGGGAGAAGGGGCTTCTCCCGCCGCTGCCGCCACCGCCGACGACGGCCGTGGC 1618 

************************************************************ 

62

KDML105_DNA CGGAGGAGACGACTCGGCGGGTGTGGCCGGCGAAAAGGAACCGATACTGGGGAGGGGGAC 1677 

BW1_DNA CGGAGGAGACGGCTCGGCGGGTGTGGCCGGCGAAAAGGAACCGATACTGGGGAGGGGGAC 1678 

Mura_DNA CGGAGGAGACGGCTCGGCGGGTGTGGCCGGCGAGAAGGAACCGATACTGGGGAGGGGGAC 1679 

Nip_DNA CGGAGGAGACGGCTCGGCGGGTGTGGCCGGCGAGAAGGAACCGATACTGGGGAGGGGGAC 1679 

Mura_cDNA CGGAGGAGACGGCTCGGCGGGTGTGGCCGGCGAGAAGGAACCGATACTGGGGAGGGGGAC 1180 

Purple_cDNA CGGAGGAGACGGCTCGGCGGGTGTGGCCGGCGAGAAGGAACCGATACTGGGGAGGGGGAC 1180 

JHN_DNA CGGAGGAGACGGCTCGACAGGTGTGGCCGGCGAGAAGGAACCGA-ATTGGGGAGGGGGAC 1676 

#3_DNA CGGAGGAGACGGCTCGGCGGGTGTGACCGGCGAGAAGGAACCGATACTGGGGAGGGGGAC 1678 

*********** **** * ****** ******* ********** * ************* 

KDML105_DNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCAAATGAGAGTTGATTAGACTGTC 1737 

BW1_DNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCAAATGGGTGTTGACTAGACTGTC 1738 

Mura_DNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCAAATGAGTGTTGACTAGTGAGTC 1739 

Nip_DNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCAAATGAGTGTTGACTAGTGAGTC 1739 

Mura_cDNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCAAATGAGTGTTGACTAGTGAGTC 1240 

Purple_cDNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCAAATGAGTGTTGACTAGTGAGTC 1240 

JHN_DNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCAAATGAGTGTTGACACGTGAGTC 1736 

#3_DNA CGGGACGGCGGTTGGTGCTGAAACAGAAGCGTTGGTCGGATGAGTGTTGACACGTGAGTC 1738 

************************************* *** * ***** * *** 

KDML105_DNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGCTGCTCCTAGCCTCGTTGGCTTCG-CTA 1796 

BW1_DNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGCTGCGCCTAGACTCGTTGGCCTCGTCTA 1798 

Mura_DNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGCTGCTCCTTGCCTCGTTGGCTTCGTCTA 1799 

Nip_DNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGCTGCTCCTTGCCTCGTTGGCTTCGTCTA 1799 

Mura_cDNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGCTGCTCCTTGCCTCGTTGGCTTCGTCTA 1300 

Purple_cDNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGCTGCGCCTTGCCTCGTTGGCTTCGTCTA 1300 

JHN_DNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGTGGCGACT-GCCTCGT-GGCTTCGTCTA 1794 

#3_DNA CAGAGAACGGTATTGAAATTGATCGTGTTTCGCTGCGCCTTGTCTAGTTGGCTTCCTCTA 1798 

******************************** ** ** * ** ** *** ** *** 

KDML105_DNA TTT-ACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGTTCATATGTAAC 1855 

BW1_DNA TTT-ACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGTTCATATGTAAC 1857 

Mura_DNA TTTCACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGTTCATATGTAAC 1859 

Nip_DNA TTTCACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGTTCATATGTAAC 1859 

Mura_cDNA TTTCACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGTTCATATGTAAC 1360 

Purple_cDNA TTTCACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGTTCATATGTAAC 1360 

JHN_DNA TTT-ACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGATCATATGTAAC 1853 

#3_DNA TTTCACAATGCGAGATTTGGAATAAATCAGAGCGGTTAATCCTGTAAGTTCATATGTAAC 1858 

*** ******************************************** *********** 

KDML105_DNA GTACCCATTTGATTTTTTATTGGTTACATATGGTTACTCCCTCCGGTTTCATTTTAATTG 1915 

BW1_DNA GTACCCATTTGATTTTTTATTGGTTACATATGGTTACTCCCTCCGGTTTCATTTTAATTG 1917 

Mura_DNA GTACCCATTTGATTTTTTATTGGTTACATATGGTTACTCCCTCCGGTTTCATTTTAATTG 1919 

Nip_DNA GTACCCATTTGATTTTTTATTGGTTACATATGGTTACTCCCTCCGGTTTCATTTTAATTG 1919 

Mura_cDNA GTACCCATTTGATTTTTTATTGGTTACATATGGTTACTCCCTCCGGTTTCATTTTAATTG 1420 

Purple_cDNA GTACCCATTTGATTTTTTATTGGTTACATATGGTTACTCCCAAAAAAAAAAAAAAAAAAA 1420 

JHN_DNA GTACCCATTGGATTTTTTATTGGTTACATATGGTTACTCCCTCCGGTTTCATTTTAATTG 1913 

#3_DNA GTACCCATTTGATTTTTTATTGGGTACATATGGTTACTCCCTCCGGTTTCATTTTAATTG 1918 

********* ************* ***************** * ** 

KDML105_DNA ACACTTTAAACAATAATACGCTTTACAAGATATACCTTTGACCTTATTTTTCTATTATAA 1975 

BW1_DNA ACACTTTGAACAATAATACGCTTTACAAGATATATCTTTGACCTTATTTTTCTATTATAA 1977 

Mura_DNA ACACTTTGAACAATAATACGTTTTACAAGATATACCTTTGACTTTATTTTTCTATTATAA 1979 

Nip_DNA ACACTTTGAACAATAATACGTTTTACAAGATATACCTTTGACTTTATTTTTCTATTATAA 1979 

Mura_cDNA ACACTTTGAACAATAATACGTTTTACAAGATATACCTTTGACTTTAAAAAAAAAAAAAAA 1480 

Purple_cDNA AA---------------------------------------------------------- 1422 

JHN_DNA ACACTTTGAACAATAATACGCTTTACAAGATATATCTTTGACCTTATTTTTCTATTATAA 1973 

#3_DNA ACACTTTGAACAATAATACGCTTTACAAGATATATCTTTGACCTTATTTTTCTATTATAA 1978 

* 

KDML105_DNA TATATACAATAAATAAATGCATGTT 2000 

BW1_DNA TATATACAATAAATAAATATATGTT 2002 

Mura_DNA TATATACAATAAATAAATGCATGTT 2004 

Nip_DNA TATATACAATAAATAAATGCATGTT 2004 

Mura_cDNA ------------------------- 

Purple_cDNA ------------------------- 

JHN_DNA TATATACAATAAATAAATATATGTT 1998 

#3_DNA TATATACAATAAATAAATATATGTT 2003 

63

Cultivated Rice shown in CLUSTAL W (1.82) 

KDML105 = Khao Dawk Mali 105 

BW1 = Jao Hom Nin mutant 1 

Mura = Murasaki 

Nip = Nipponbare 

Purple = Purple Puttu 

JNN = Jao Hom Nin 

#3 = Jao Hom Nin number 3 

64

THESIS

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