ORIGINAL PAPER

Gain of deleterious function causes an autoimmune responseand Bateson–Dobzhansky–Muller incompatibility in rice

Eiji Yamamoto • Tomonori Takashi • Yoichi Morinaka • Shaoyang Lin •

Jianzhong Wu • Takashi Matsumoto • Hidemi Kitano • Makoto Matsuoka •

Motoyuki Ashikari

Received: 7 October 2009 / Accepted: 18 January 2010 / Published online: 6 February 2010

� Springer-Verlag 2010

Abstract Reproductive isolation plays an important role

in speciation as it restricts gene flow and accelerates

genetic divergence between formerly interbreeding popu-

lation. In rice, hybrid breakdown is a common reproductive

isolation observed in both intra and inter-specific crosses. It

is a type of post-zygotic reproductive isolation in which

sterility and weakness are manifested in the F2 and later

generations. In this study, the physiological and molecular

basis of hybrid breakdown caused by two recessive genes,

hbd2 and hbd3, in a cross between japonica variety,

Koshihikari, and indica variety, Habataki, were investi-

gated. Fine mapping of hbd2 resulted in the identification

of the causal gene as casein kinase I (CKI1). Further

analysis revealed that hbd2-CKI1 allele gains its deleteri-

ous function that causes the weakness phenotype by a

change of one amino acid. As for the other gene, hbd3 was

mapped to the NBS-LRR gene cluster region. It is the most

common class of R-gene that triggers the immune signal in

response to pathogen attack. Expression analysis of

pathogen response marker genes suggested that weakness

phenotype in this hybrid breakdown can be attributed to an

autoimmune response. So far, this is the first evidence

linking autoimmune response to post-zygotic isolation in

rice. This finding provides a new insight in understanding

the molecular and evolutionary mechanisms establishing

post-zygotic isolation in plants.

Keywords Rice � Reproductive isolation �BDM incompatibility � Autoimmune response �Weakness phenotype

Introduction

Reproductive isolation contributes to speciation by pre-

venting or restricting gene exchange between and within

species (Coyne and Orr 2004; Rieseberg and Willis 2007).

Based on the mode of action, reproductive isolation can be

classified into two main types: pre-zygotic and post-zygotic

isolation. Of the two, pre-zygotic isolation is most common

and prevents both inter and intra-specific crosses through

geographical isolation, difference in flowering time, polli-

nator specificity, incompatibility in pollen tube growth, etc.

(Stebbins 1950). In contrast, post-zygotic isolation appears

after the zygotes or hybrids are developed. It is often

manifested in the hybrids as embryonic lethality, seed

inviability, weakness, and sterility. This reproductive iso-

lation renders the species or populations genetically

incompatible and contributes significantly to both animal

and plant speciation (Rieseberg et al. 2006).

Among the subtypes of post-zygotic isolations (Stebbins

1950), ‘‘hybrid sterility’’ and ‘‘hybrid weakness’’ (also

called inviability or necrosis) are observed in F1 hybrids,

whereas ‘‘hybrid breakdown’’ is manifested in the F2 or

Communicated by K. Shirasu.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00438-010-0514-y) contains supplementarymaterial, which is available to authorized users.

E. Yamamoto � H. Kitano � M. Matsuoka � M. Ashikari (&)

Bioscience and Biotechnology Center, Nagoya University,

Nagoya 464-8601, Japan

e-mail: ashi@agr.nagoya-u.ac.jp

T. Takashi � Y. Morinaka � S. Lin

Honda Research Institute Japan, Kazusa-Kamatari,

Kisarazu, Chiba 292-0818, Japan

J. Wu � T. Matsumoto

National Institute of Agrobiological Resources,

Tsukuba 305-8602, Japan

123

Mol Genet Genomics (2010) 283:305–315

DOI 10.1007/s00438-010-0514-y

later generations through sterility or weakness phenotype.

The genetic mechanisms of post-zygotic isolation are

theoretically explained by Bateson–Dobzhansky–Muller

(BDM) model (Dobzhansky 1937; Muller 1942; Coyne and

Orr 2004), which postulates that deleterious interaction of

two or more genes derived from different species or pop-

ulation causes post-zygotic isolation.

So far, few reports have identified BDM incompatibility

genes and their underlying molecular mechanisms in plant.

In Arabidopsis, the complementary loss of duplicated

genes had been shown to cause embryonic lethality in the

F2 progenies of intra-specific cross (Bikard et al. 2009).

Two other investigations have identified the causal genes

of hybrid sterility in rice (Oryza sativa L.) (Chen et al.

2008; Long et al. 2008).

The weakness phenotype is observed in both ‘‘hybrid

weakness’’ and ‘‘hybrid breakdown’’ and considered a

common type of post-zygotic isolation in plants (Bomblies

and Weigel 2007). However, weakness phenotype has

received less attention compared with other phenotypes,

such as sterility and inviability. In Arabidopsis, an auto-

immune response was reported to be involved in the

weakness of hybrids (Bomblies et al. 2007; Alcazar et al.

2009). But in other plants, the physiological and molecular

mechanisms for the appearance of weakness phenotype

remain to be established. Rice has great potential to

investigate and understand this phenomenon as weakness

phenotype is observed in numerous intra and inter-specific

crosses (Amemiya and Akamine 1963; Sato and Morishima

1988; Fukuoka et al. 1998, 2005; Kubo and Yoshimura

2002; Matsubara et al. 2007; Yamamoto et al. 2007; Miura

et al. 2008).

Previously, we reported a hybrid breakdown involving

weakness phenotype in the F2 progenies of Koshihikari

(O. sativa L. ssp. japonica) and Habataki (O. sativa L. ssp.

indica; Yamamoto et al. 2007). Genetic analysis revealed

this hybrid breakdown to be controlled by two recessive

genes: hybrid breakdown 2 (hbd2) and hbd3. Plant carrying

a chromosomal segment from the hbd2 region of Habataki

and the hbd3 region of Koshihikari exhibits weakness

phenotype (Fig. 1c), unlike the parent cultivars (Fig. 1a) or

plants heterozygous for hbd2 or hbd3 (Fig. 1b, d). In this

study, the molecular and physiological mechanisms by

which these two genes interact causing the weakness

phenotype were investigated.

Materials and methods

Plant materials

Koshihikari (O. sativa L. ssp. japonica) and a series of

nearly isogenic lines (NILs) were used in this study

(Fig. 1). The methods for the construction of NILs and the

hbd2/hbd2

Hbd3/hbd3

hbd2/hbd2

hbd3/hbd3

Hbd2/hbd2

hbd3/hbd31 2 3 4 5 6 7 9 10 11 1281 2 3 4 5 6 7 9 10 11 1281 2 3 4 5 6 7 9 10 11 1281 2 3 4 5 6 7 9 10 11 128

Hbd2/Hbd2

hbd3/hbd3Koshihikari NIL-Hbd2/hbd2 + hbd3/hbd3 NIL-hbd2/hbd2 + hbd3/hbd3 NIL-hbd2/hbd2 + Hbd3/hbd3

a b c d

Fig. 1 Morphology and graphical genotype of Koshihikari (Hbd2/Hbd2 ? hbd3/hbd3), NIL-Hbd2/hbd2 ? hbd3/hbd3, NIL-hbd2/hbd2 ? hbd3/hbd3 and NIL-hbd2/hbd2 ? Hbd3/hbd3 plants. Only

NIL with the genotype hbd2/hbd2 ? hbd3/hbd3 exhibits the

weakness phenotype. Horizontal lines in graphical genotypes indicate

the position of markers used for genotyping. Yellow and blue zonesindicate the Koshihikari and Habataki chromosome, respectively.

Scale bar 1 m

306 Mol Genet Genomics (2010) 283:305–315

123

molecular markers used for genotyping were as described

in Yamamoto et al. (2007).

Fine-scale mapping of hbd2 and hbd3

For fine-scale mapping, 11,520 progenies of NIL-Hbd2/

hbd2 ? hbd3/hbd3 and 5,760 progenies of NIL-hbd2/

hbd2 ? Hbd3/hbd3 were analyzed for the hbd2 and hbd3,

respectively. The genomic DNA of the progenies

was extracted using the TPS (Tris–Potassium chloride–

Disodium salt) method for genotyping and screening

fine-scale recombinants, as follows. For the TPS method,

approximately 2 cm lengths rice leaf tips were harvested and

ground using a Multi-Beads Shocker (Yasui Kikai, Osaka,

Japan) in TPS buffer [100 mM Tris–HCl (pH 8.0), 1 M KCl,

and 10 mM EDTA]. After centrifuging, the supernatant was

recovered and an equal volume of isopropyl alcohol was

added. Isopropyl alcohol-insoluble material was recovered

by centrifugation, and the pellet was washed with 75%

ethanol. Thereafter, the pellet was dried and dissolved in TE

[10 mM Tris–HCl (pH 8.0) and 1 mM EDTA]. Primer

sequences of the molecular markers used for genotyping are

shown in Supplementary Table 1.

DNA sequencing and gene prediction of candidate

regions

Koshihikari genome sequencing was performed using

Illumina Genome Analyzer system (Illumina, San Diego,

CA, USA) according to manufacturer’s specifications.

Derived sequences were then assembled using maq (http://

maq.sourceforge.net/). In the Habataki sequence, BAC

clone HAB027I16 was isolated for hbd2 while the BAC

clones HAB024P12, Haba40C09, and HAB046J05 were

isolated for hbd3. Gene prediction in the hbd2 and hbd3

candidate region was performed using the Rice Genome

Automated Annotation System (http://RiceGAAS.dna.

affrc.go.jp/) and Eukaryotic GeneMark.hmm (Lomsadze

et al. 2005).

DNA construction and rice transformation

To produce transgenic plants over-expressing Koshihikari

or Habataki CKI1, the coding sequences were amplified by

RT-PCR using the primer pair 50-TCT AGA ATG GAG

CAT GTG ATC GGG-30 and 50-CCC GGG TTA TTT CCT

TCT GTC AGC A-30. Amplified fragments were intro-

duced into pCRII (Invitrogen, Carlsbad, CA, USA). The

clones were sequenced to confirm that no base substitutions

had occurred during PCR. Cloned fragments were digested

with XbaI and SmaI (TaKaRa, Otsu, Japan), introduced

into the binary vector pNiR::NiRcDNA::NiRt and trans-

formed in Koshihikari calli, as described by Nishimura

et al. (2005). The expression of both CKI1 s was controlled

by the rice Actin promoter from pBI-Hm2.

Expression analysis

Total RNA was extracted using the RNeasy plant kit

(Qiagen, Venlo, The Netherlands). First-strand cDNA was

then synthesized from about 1 lg of total RNA using an

oligo(dT) primer and Omniscript RT kit (Qiagen). Quanti-

tative real-time PCR was conducted using the LightCycler

system (Roche, Basel, Switzerland) and the QuantiTect

SYBR Green PCR kit (Qiagen). For this analysis, a linear

standard curve and threshold cycle number versus log

(designated transcript level) were constructed using a series

of dilutions of each PCR product (10-17, 10-18, 10-19, and

10-20M); subsequently, the levels of transcript in all

unknown samples were determined by the standard curve.

UBQ1 was used as an internal standard for normalizing

cDNA concentration variations. Data represent the average

of five replicates. The sequences of primer pairs are

described in Supplementary Table 2.

Distribution analysis of hbd2 mutation

The distribution of the hbd2 mutation was analyzed by

sequencing. The primer pair 50-CGG AGA GCA CAC

AAA GCA CAG-30 and 50-TCC AGA ATA CAG AGT

TCC AGC-30 was used to amplify the hbd2 mutation site in

Koshihikari, Habataki, 69 accessions in the World Rice

Collection (Kojima et al. 2005), and several ancestral lines

of Milyang 23. DNA sample of Peta and IR262 were kindly

provided by the International Rice Research Institute.

Results

Fine-scale mapping and function of hbd2

hbd2 is one of the recessive genes involved in the hybrid

breakdown observed in the cross progenies of Koshihikari

and Habataki. The gene is located on the long arm of

chromosome 2 (Figs. 1c, 2a; Yamamoto et al. 2007).

NIL-hbd2/hbd2 ? hbd3/hbd3, carrying the homozygous

alleles of Habataki hbd2 locus in the Koshikari genetic

background, exhibits the weakness phenotype compared to

Koshihikari (Hbd2/Hbd2 ? hbd3/hbd3).

Fine-scale mapping of hbd2 was performed using

11,520 progenies of NIL-Hbd2/hbd2 ? hbd3/hbd3

(Fig. 1b). This resulted to the narrowing of the hbd2 can-

didate region to a distance of 17 kb located between two

molecular markers, dj1a-F3 and dj1a-F7 (Fig. 2b). Gene

prediction revealed that this region contains only one open

reading frame, which encodes casein kinase I (CKI1)

Mol Genet Genomics (2010) 283:305–315 307

123

(Fig. 2c). CKI1 consists of 14 exons and encodes 463

amino acids. Although a comparison of DNA sequences

revealed the presence of many mutations between Koshi-

hikari and Habataki in this genomic region, only one amino

acid change was observed in CKI1 (Fig. 2c, d). The amino

acid substitution occurred in the variable domain (Fig. 2d)

(Knippschild et al. 2005) with amino acid 357 changing

from isoleucine in Koshihikari to lysine in Habataki.

Quantitative RT-PCR was performed to analyze the

level of CKI1 transcripts in the different tissues of Koshi-

hikari. CKI1 transcripts were detected in the leaf, stem,

vegetative shoot apex, flower, and root (Fig. 2e). The leaf,

however, showed the highest transcripts level compared

with the other tissues. We also compared the level of CKI1

transcripts in leaves with different genetic or phenotypic

background (Fig. 2f). The leaves of Koshihikari

Centromerehbd2

2S 2L

2 2 21 4

dj1a

-61

dj1a

-F1

dj1a

-F3

dj1a

-F7

dj1a

-F8

dj1a

-16

17 kb

a

b

c

1 kb :

d

Protein Kinase Domain Variable Domain

I 357 K

g

Koshih

ikari

NIL-h

bd2/

hbd2

KoCKI1

HaCKI1

Empt

y vec

tor

over

-exp

ress

er

over

-exp

ress

er

A ATAAA

Ko : Ha :

IleLys

20

16

12

8

4

0L1 L2 L3 L4 L5 L6 L7

Empt

y vec

tor

f

Koshih

ikari

NIL-h

bd2/

hbd2

+ Hbd

3/Hbd

3

NIL-h

bd2/

hbd2

x1.0 x1.2 x1.2x2.9

x4.5

x11.3 x11.7x13.4 x13.5

x18.8

x1.0

e40

30

20

10

0

x33.4

x1.0 x1.2

x5.6 x4.5

Leaf

Stem

shoo

t ape

x

Flower

Shoot

ape

xRoo

t

Veget

ative

Repro

ducti

ve

+ hb

d3/h

bd3

+ hb

d3/h

bd3

Fig. 2 Cloning of hbd2. a Location of hbd2 on chromosome 2.

b High-resolution linkage map of hbd2. Red arrow indicates the

position of hbd2 while the vertical bars represent the position of

molecular markers. The number of recombinants between the

molecular markers is indicated between the vertical bars. c Primary

structure of CKI1 gene. CKI1 corresponds to Os02g0622100 in the

RAP2 loci. Black boxes represent the coding sequence. Gray box on

the left represents the 50-UTR, and the grey pentagon on the right

represents the 30-UTR. Arrow indicates the position of the SNP that

causes the amino acid change in CKI1 protein. d Primary structure of

the CKI1 protein. Arrow indicates the position of the amino acid

change. Amino acid 357 is changed from isoleucine in Koshihikari to

lysine in Habataki. e Quantitative RT-PCR analysis of tissue specific

expression of CKI1 in Koshihikari. Bars represent the mean ± SD.

UBQ1 was used as an internal standard for normalization. fQuantitative RT-PCR analysis of CKI1 in Koshihikari (Hbd2/Hbd2 ? hbd3/hbd3), NIL-hbd2/hbd2 ? hbd3/hbd3, NIL-hbd2/hbd2 ? Hbd3/Hbd3 and Habataki CKI1 over-expressers. L1–L7 are

transgenic plants over-expressing Habataki CKI1. Bars mean ± SD.

UBQ1 was used as an internal standard for normalization. g Plant

morphology of Koshihikari (Hbd2/Hbd2 ? hbd3/hbd3), NIL-hbd2/hbd2 ? hbd3/hbd3, Koshihikari CKI1 over-expresser, Habataki CKI1

over-expresser and empty vector control plant. KoCKI1 and HaCKI1indicate Koshihikari and Habataki CKI1 over-expresser, respectively.

Both CKI1 coding sequences are under the control of rice Actinpromoter. Scale bar 1 m

308 Mol Genet Genomics (2010) 283:305–315

123

(Hbd2/Hbd2 ? hbd3/hbd3), NIL-hbd2/hbd2 ? hbd3/hbd3

and NIL-hbd2/hbd2 ? Hbd3/Hbd3 were used in this

analysis. It appears that CKI1 transcripts in plants homo-

zygous for hbd2 allele is higher than the plant homozygous

for the dominant Hbd2 allele (Koshihikari), but the dif-

ference was not significant (Fig. 2f). Similarly, plant which

shows the weakness phenotype (NIL-hbd2/hbd2 ? hbd3/

hbd3) did not show significant difference in transcripts

level of CKI1 compared with the normal growth NIL-hbd2/

hbd2 ? Hbd3/Hbd3 (Fig. 2f).

We tried to confirm CKI1 as the causal gene of hbd2 by

transforming the Koshihikari CKI1 genomic region into

NIL-hbd2/hbd2 ? hbd3/hbd3 calli. No regenerants, how-

ever, were obtained from NIL-hbd2/hbd2 ? hbd3/hbd3

calli.

As an alternative to the above-mentioned strategy, the

Koshihikari and Habataki CKI1 coding sequences were

placed under the control of the rice Actin promoter and

were transformed into Koshihikari calli to produce CKI1

over-expressers (Fig. 2g). In contrast with NIL-hbd2/

hbd2 ? hbd3/hbd3 calli, Koshihikari calli were easily

regenerated. Koshihikari CKI1 over-expressers showed a

significant increase in CKI1 transcripts compared with

Koshihikari, NIL-hbd2/hbd2 ? hbd3/hbd3 and NIL-hbd2/

hbd2 ? Hbd3/Hbd3 (data not shown). All of them, how-

ever, showed normal phenotype similar to Koshihikari.

Habataki CKI1 over-expressers also showed varying but

significantly higher CKI1 transcript levels than Koshihik-

ari, NIL-hbd2/hbd2 ? hbd3/hbd3 and NIL-hbd2/

hbd2 ? Hbd3/Hbd3 (Fig. 2f). Unlike the Koshihikari CKI1

over-expressers, all the Habataki CKI1 over-expressers

showed weakness phenotype very similar to that of NIL-

hbd2/hbd2 ? hbd3/hbd3 (Fig. 2g). In fact, the morpho-

logical characteristics of Habataki CKI1 over-expressers

did not differ significantly from that of NIL-hbd2/

hbd2 ? hbd3/hbd3 (Table 1). These results indicate that

the existence of Habataki CKI1 in the genetic background

of Koshihikari causes the weakness phenotype. Thus, CKI1

is determined as the causal gene of hbd2. Furthermore, the

single amino acid change in the CKI1 of Koshihikari and

Habataki appeared to be the causal mutation. This involves

the change of amino acid 357 from isoleucine in Koshi-

hikari to lysine in Habataki.

Fine-scale mapping of hbd3

The recessive gene hbd3 interacts with hbd2 to cause the

weakness phenotype. Its dominant allele, Hbd3, found in

the Habataki genome represses the deleterious effect of

hbd2 (Fig. 1d; Yamamoto et al. 2007). hbd3 is located on

the long arm of chromosome 11 (Fig. 3a; Yamamoto et al.

2007). Fine-scale mapping of the gene using 5,760 prog-

enies from NIL-hbd2/hbd2 ? Hbd3/hbd3 (Fig. 1d) resul-

ted in the narrowing of the candidate region to 168.7 kb in

Koshihikari and 130.4 kb in Habataki (Fig. 3c). These

regions are located between the molecular markers dj1b-12

and dj1b-20 (Fig. 3b). A DNA sequence comparison of the

two target regions revealed that both sequences are highly

diversified, with no sequence similarities in most of the

regions (Fig. 3c). Gene prediction of the two sequences,

however, showed that both sequences posses the NBS-LRR

gene cluster (Fig. 3c), which is the most common class of

disease resistance (R) genes in plants (Jones and Dangl

2006). Some predicted genes in the hbd3 candidate regions

contained both nucleotide binding site (NBS) and leucine

rich repeat (LRR) domains (Fig. 3c, red pentagons), while

others have only NBS (Fig. 3c, orange pentagon) or LRR

(Fig. 3c, yellow pentagon) domains.

Expression analysis of pathogen response marker genes

NBS-LRR triggers the immune response signal in response

to a pathogen attack (Chisholm et al. 2006; DeYoung and

Innes 2006). In Arabidopsis, Bomblies et al. (2007) and

Alcazar et al. (2009) indicated that NBS-LRR is involved in

weakness of hybrid and demonstrated the activation of the

immune response signal in weakness phenotype. As hbd3

was mapped to the NBS-LRR gene cluster, we suspected

that an autoimmune response is responsible for the weak-

ness phenotype. To confirm this hypothesis, the expression

level of pathogen response marker genes in Koshihikari,

NIL-hbd2/hbd2 ? hbd3/hbd3, and Habataki CKI1 over-

expresser were examined using quantitative RT-PCR

(Fig. 4a). Pathogen response marker genes are known to be

up-regulated by the activation of immune response signal.

Without inoculation of any pathogen, most of the analyzed

pathogen response marker genes were up-regulated in

NIL-hbd2/hbd2 ? hbd3/hbd3 and Habataki CKI1 over-

expresser as compared with Koshihikari (Fig. 4b). These

results suggest that the immune response signal is activated

in NIL-hbd2/hbd2 ? hbd3/hbd3 and Habataki CKI1 over-

expresser. High cell metabolism required in maintaining an

Table 1 Plant height and tiller number of Koshihikari, NIL-hbd2/hbd2 ? hbd3/hbd3, KoCKI1 and HaCKI1 over-expresser and control

plant

Line Plant height (cm) Number of tillers

Koshihikari 120.5 ± 3.5 11.6 ± 2.1

NIL-hbd2 ? hbd3/hbd3 86.4 ± 5.5 4.3 ± 0.6

KoCKI1 over-expresser 118.5 ± 4.4 12.0 ± 1.9

HaCKI1 over-expresser 82.6 ± 6.2 3.7 ± 1.6

Empty vector control 120.2 ± 2.6 12.3 ± 2.5

The numbers represent the average from seven plants

KoCKI1 and HaCKI1 indicate Koshihikari and Habataki CKI1,

respectively

Mol Genet Genomics (2010) 283:305–315 309

123

activated immune response signal has been reported to

reduce plant growth (Tian et al. 2003; van Hulten et al.

2006). Thus, the weakness phenotype observed in rice

hybrid breakdown can be attributed to an autoimmune

response.

Origin of the hbd2 mutation

In this study, we succeeded in determining the causal

mutation of hbd2. The distribution of hbd2 mutation in rice

was surveyed using 69 accessions from the World Rice

Collection (Kojima et al. 2005). Each of the accessions was

sequenced for the mutation site of hbd2. Of the 69 acces-

sions surveyed, only Milyang 23 possessed the hbd2

mutation. Both Habataki and Milyang 23 are indica type

modern varieties developed in recent breeding programs.

As the genealogy of Habataki is too complicated, only the

genealogy of Milyang 23, was considered for further

analysis to identify the parent donor of hbd2 (Fig. 5a). The

ancestral parents of Milyang 23 were surveyed for the hbd2

genotype and found that hbd2 mutation was inherited from

Peta, an indica type landrace from Indonesia (Fig. 5b).

Apparently, the hbd2 mutation has occurred naturally in

Peta without deleterious effect. It was then conserved

neutrally in the cultivar and by chance incorporated into

few modern varieties through the rice breeding programs.

Discussion

Post-zygotic isolation contributes significantly to plant

speciation (Rieseberg et al. 2006). Knowledge of the

molecular and physiological mechanism involved in this

reproductive isolation, therefore, is important to better

understand of plant speciation. In a previous study, we

reported a hybrid breakdown with the weakness phenotype

in F2 progenies of Koshihikari (O. sativa L. ssp. japonica)

and Habataki (O. sativa L. ssp. indica; Yamamoto et al.

2007). Hybrid breakdown is a sub-type of post-zygotic

isolation observed in F2 or later generations. Genetic

analysis revealed that this hybrid breakdown is controlled

by two recessive genes, hbd2 and hbd3. Plant carrying a

Centromerehbd3

11S 11L

dj1b

-22

dj1b

-12

dj1b

-20

dj1b

-15

dj1b

-11

126 3 0 5

a

b

dj1b

-23

Koshihiikari : 168.7 kb

Habataki : 130.4 kb

Ko

Ha

dj1b

-12

dj1b

-11

dj1b

-23

dj1b

-20

79000 79100 79232 79300LOC

79500 80000 81000 81100 81200 8145081500

81550

816008180081900

c

Fig. 3 Fine-scale mapping of hbd3. a Location of hbd3 on chromo-

some 11. b High-resolution linkage map of hbd3. Red arrow shows

the position of hbd3 while the vertical bars represent the position of

molecular markers. The number of recombinants between molecular

markers is indicated between the vertical bars. c Comparison of the

genetic structure in the hbd3 candidate region between Koshihikari

and Habataki. Ko and Ha indicate the Koshihikari and Habataki

sequences, respectively. Red pentagons denote NBS-LRR gene

clusters. Orange pentagons show the coding sequences of only the

NBS domain. Yellow pentagons indicate the coding sequences of only

the LRR domain. Light green pentagons denote the coding sequences

of other gene classes. Gray pentagons indicate hypothetical proteins.

Black pentagons indicate transposable elements. Genes related to

NBS-LRR are enclosed by bold lines. The five digits next to the genes

identify the last corresponding RAP2 loci (Os11g04XXXXX). LOC

corresponds to MSU Osa1 Rice Loci LOC Os11g29000. Blue shadedregion connecting the two sequences represent the region where DNA

sequence similarity are recognized. Vertical bars show the position of

molecular markers

310 Mol Genet Genomics (2010) 283:305–315

123

chromosomal segment from the hbd2 region of Habataki

and hbd3 region of Koshihikari shows the weakness phe-

notype (Fig. 1c). In this study, we determined that hbd2

encodes CKI1 and one amino acid substitution is the causal

mutation for the hybrid breakdown. On the other hand,

fine-scale mapping revealed that hbd3 maps to NBS-LRR

gene cluster which is the most common class of disease

resistance (R) gene in plants. Because NBS-LRRs trigger

immune response, we suspected the involvement of

immune response signal in this hybrid breakdown. The

result of expression analysis of pathogen response marker

genes supports this idea. Involvement of autoimmune

response in weakness of hybrid has been already reported

in Arabidopsis (Bomblies et al. 2007; Alcazar et al. 2009).

Our results suggest that this mechanism also present in rice.

This also implies that this kind of mechanism has greater

potential for the establishment of post-zygotic isolation

with weakness phenotype in plants.

hbd2 encodes CKI1 and gains deleterious function to

cause weakness phenotype

To establish the mechanism of hybrid breakdown in this

study, we identified the causal gene of hbd2. Fine-scale

mapping of hbd2 identified only one open reading frame,

which encodes CKI1 (Fig. 2c). In a comparison of amino

PR1a PR1b

Lipoxygenase

Koshih

ikari

NIL-h

bd2/

hbd2

HaCKI1

over

-exp

ress

er

Koshih

ikari

NIL-h

bd2/

hbd2

Koshih

ikari

NIL-h

bd2/

hbd2

0

1.2

1.6

2.0

0.8

0.4

PBZ1

Koshih

ikari

NIL-h

bd2/

hbd2

0

8.0

10.0

6.0

4.0

2.0

0

7.0

5.0

3.0

1.0

0

1.0

2.0

3.0

4.0

5.0

2.0

4.0

6.0

HaCKI1

over

-exp

ress

er

HaCKI1

over

-exp

ress

er

HaCKI1

over

-exp

ress

er

Rel

ativ

e m

RN

A le

vel

Rel

ativ

e m

RN

A le

vel

Rel

ativ

e m

RN

A le

vel

Rel

ativ

e m

RN

A le

vel

0

1.0

2.0

3.0

4.04.5

Koshih

ikari

NIL-h

bd2/

hbd2

HaCKI1

over

-exp

ress

er

Rel

ativ

e m

RN

A le

vel GST-u4

0

1.0

2.0

3.0

4.0

6.0

Rel

ativ

e m

RN

A le

vel

5.0

Koshih

ikari

NIL-h

bd2/

hbd2

HaCKI1

over

-exp

ress

er

Cytochrome P450

0

8.0

10.0

6.0

4.0

2.0

Rel

ativ

e m

RN

A le

vel

Koshih

ikari

NIL-h

bd2/

hbd2

HaCKI1

over

-exp

ress

er

PR4

Koshih

ikari

NIL-h

bd2/

hbd2

HaCKI1

over

-exp

ress

er

PR2

0

0.4

0.8

1.2

1.6

Rel

ativ

e m

RN

A le

vel

Gene Locus ID References

PR1a Os07g0129200 Agrawal et al. 2000a

PR1b Os01g0382000 Agrawal et al. 2000b

PR2 Os01g0940700 Shimono et al. 2007

PR4 Os11g0592200 Agrawal et al. 2003

GST-u4 Os10g0528300 Shimono et al. 2007

Cytochrome 450 Os07g0418500 Shimono et al. 2007

PBZ1 Os12g0555500 Midoh and Iwata 1996

Lipoxygenase Os12g0559200 Schaffrath et al. 2000

a

b

+ hb

d3/h

bd3

+ hb

d3/h

bd3

+ hb

d3/h

bd3

+ hb

d3/h

bd3

+ hb

d3/h

bd3

+ hb

d3/h

bd3

+ hb

d3/h

bd3

+ hb

d3/h

bd3

Fig. 4 Expression analysis of pathogen response marker genes. a List

of marker genes for pathogen response used in this study. Sequence

data of the genes are in the Rice Annotation Project Database

(RAP-DB). b Quantitative RT-PCR analysis of marker genes for

pathogen response in Koshihikari (Hbd2/Hbd2 ? hbd3/hbd3),

NIL-hbd2/hbd2 ? hbd3/hbd3 and Habataki CKI1 over-expresser.

Total RNAs were extracted from the fourth leaf of sixth-leaf stage

plants and reverse transcribed to obtain cDNA. Bars represent the

means ± SD. UBQ1 was used as an internal standard for normali-

zation. HaCKI1 indicates Habataki CKI1 over-expresser

Mol Genet Genomics (2010) 283:305–315 311

123

acid sequences, we found only one amino acid change in a

variable domain that is known to be less conserved in this

family (Fig. 2d; Gross and Anderson 1998; Knippschild

et al. 2005). Because hbd2 is a recessive gene, we initially

tried to rescue the NIL-hbd2/hbd2 ? hbd3/hbd3’s weak-

ness phenotype by transforming the Koshihikari CKI1

sequence. However, we could not produce any regenerants

from NIL-hbd2/hbd2 ? hbd3/hbd3 calli through Agro-

bacterium-mediated transformation for unknown reasons.

As an alternative strategy, we transformed the Habataki

CKI1 coding sequence under the control of the rice Actin

promoter into Koshihikari calli. This experiment was very

successful, with the phenotype of the Habataki CKI1 over-

expresser resembling that of NIL-hbd2/hbd2 ? hbd3/hbd3

(Fig. 2g; Table 1). This result indicates that Habataki CKI1

exerts the deleterious effect causing the weakness pheno-

type in the Koshihikari genetic background (Hbd2/

Hbd2 ? hbd3/hbd3). We can rule out the possibility of a

deleterious effect of CKI1 over-expression, as the Koshi-

hikari CKI1 over-expresser did not show the weakness

phenotype (Fig. 2g; Table 1). However, this result is unu-

sual. If the presence of Habataki CKI1 is deleterious in the

Koshihikari genetic background, heterozygous (Hbd2/

hbd2) plant should show the weakness phenotype. How-

ever, hbd2 is a complete recessive gene, and NIL-Hbd2/

hbd2 ? hbd3/hbd3 shows a normal growth phenotype

(Fig. 1b). This contradicts the results of the Habataki CKI1

over-expresser experiment. One explanation is that

Koshihikari CKI1 is completely replaced by over-expres-

sed Habataki CKI1 in the over-expresser. However, we

think this is unlikely, as our analysis of the relative quantity

of CKI1 transcripts in the Habataki CKI1 over-expresser

showed about 10–50% to be Koshihikari type CKI1 (data

not shown). Alternatively, we suggest that this weakness

phenotype is determined by the quantity of Koshihiakri or

Habataki CKI1. hbd2/hbd2 is most likely the only genotype

that makes sufficient quantity of CKI1 to cause the weak-

ness phenotype.

Another question to answer is which CKI1 determines

the weakness phenotype? If Habataki CKI1 had a loss-of-

function allele, such as kinase activity or substrate speci-

ficity loss, and if the amount of normally functioning

Koshihikari CKI1 was reduced in Habataki CKI1 over-

expresser, the reduced amount of Koshihikari CKI1 would

be the determinant of the weakness phenotype. To inves-

tigate this hypothesis, we produced transgenic lines with a

reduced CKI1 transcription level due to over-expression of

the CKI1 antisense strand. However, no phenotypic

abnormality was observed in any of the transgenic plants

(data not shown). Although we cannot exclude the possi-

bility that the amount of CKI1 was insufficiently reduced,

and it requires further analyses such as the observation of

the CKI1 loss-of-function mutant, we suggest that Koshi-

hikari CKI1 quantity does not determine the weakness

phenotype. Moreover, larger amounts of Habataki CKI1

appear to be the determinant of this weakness phenotype,

and our experiments produced no evidence to the contrary.

Thus, we concluded that hbd2 encodes CKI1 and that larger

amounts of hbd2-CKI1 with one amino acid change in the

variable domain causes the weakness phenotype in

Koshihikari genetic background.

hbd3 maps to NBS-LRR gene cluster

The other gene involved in hybrid breakdown investigated

in this study is hbd3. Fine-scale mapping failed to identify

the causal gene of hbd3, as we could not narrow down the

candidate region of the gene to less than 168.7 kb in

Koshihikari and 130.4 kb in Habataki due to difficulty in

obtaining fine-scale recombinants (Fig. 3b). The reason for

this became clear after the DNA sequences from both

TN1

Peta

Norin 13

Hutaba

Dee-geo

Peta

CP231

SL0-17

Sigadis

IR262

IR8

IR127-2

Shinkoh

IR24

Milyang 23

-woo-gen

Suwon232

Line name Type Category Origin Genotype

Norin 13 Japonica Variety Japan Hbd2

Hutaba Japonica Variety Japan Hbd2

Peta Indica Landrace Indonesia hbd2

TN1 Indica Variety Taiwan Hbd2

Dee-geo-woo-gen Indica Landrace Taiwan Hbd2

CP231 Japonica Variety U.S.A. N.A.

SLO-17 Indica Variety ? N.A.

Sigadis Indica Variety Indonesia N.A.

Shinkoh Japonica Variety Japan N.A.

IR262 Indica Variety Philippines hbd2

IR8 Indica Variety Philippines hbd2

IR127-2 Indica Variety Philippines N.A.

Suwon 232 Japonica/Indica Variety Korea hbd2

IR24 Indica Variety Philippines Hbd2

Milyang 232 Japonica/Indica Variety Korea hbd2

a

b

Fig. 5 Origin of the hbd2 mutation. a Genealogy of Milyang 23.

Lines with hbd2 mutation are enclosed in red squares. b List of

varieties and landraces in the genealogy of Milyang 23. NA not

analyzed for hbd2

312 Mol Genet Genomics (2010) 283:305–315

123

candidate regions were compared and found to be highly

diversified to recombine. Gene prediction revealed that

both candidate regions posses the NBS-LRR gene cluster

(Fig. 3c), where highly diversified sequences are often

observed (Bergelson et al. 2001). NBS-LRR genes are the

most common class of disease resistance (R) genes that

trigger an immune response signal upon recognition of

pathogen attack (Jones and Dangl 2006). Although other

gene classes exist in hbd3, we suppose that one or more

NBS-LRR genes can be the causal gene of hbd3, as two

previous reports of Arabidopsis hybrid weakness mapped

the causal gene to the NBS-LRR gene cluster (Bomblies

et al. 2007; Alcazar et al. 2009). Moreover, the phenotypic

similarity (i.e. weakness) and circumstances in our fine-

scale mapping results, indicate that the hybrid breakdown

of this study is also caused by NBS-LRR(s).

Generally, mutations in NBS-LRR which cause auto-

immune response behave dominantly. hbd3 in Koshihikari,

however, is a recessive gene and contradicts to the spec-

ulation that hbd3 encodes NBS-LRR(s). RPM1, one of the

best characterized NBS-LRR in Arabidopsis, cause dose

dependent weakness phenotype in the background

of reduced RPM1-interacting protein 4 (RIN4) level

(Belkhadir et al. 2004). This suggests that, in some situa-

tion, activity of immune response signal triggered by

NBS-LRR(s) is affected by the number of alleles. Actually,

Arabidopsis hybrid weakness gene DM1 which encodes

NBS-LRR behave dominantly, but homozygous alleles of

the gene causes stronger phenotype than heterozygote

(Bomblies et al. 2007). It is possible, therefore, that in this

hybrid breakdown, two copies not one (homozygous) of

NBS-LRR in hbd3 are needed to cause detectable immune

response.

Pathogen response marker genes are up-regulated in

weakness phenotype

Based on the result of fine-scale mapping of hbd3, we

speculate that an immune response signal is activated in

hybrid breakdown in rice, as in Arabidopsis hybrid weak-

ness (Bomblies et al. 2007; Alcazar et al. 2009). To con-

firm this idea, we conducted expression analysis of several

molecular marker genes for pathogen response (Fig. 4a).

Most of the genes analyzed were up-regulated in NIL-

hbd2/hbd2 ? hbd3/hbd3 and Habataki CKI1 over-expres-

ser compared with Koshihikari (Fig. 4b). In addition, the

two expression profiles were very similar, indicating that

Habataki CKI1 over-expresser completely mimics the

phenotype of NIL-hbd2/hbd2 ? hbd3/hbd3, not only in

morphological characteristics (Table 1) but also in the

physiological mechanism. These results support our

hypothesis that NBS-LRR(s) comprise the causal gene of

hbd3. Although significant up-regulation was observed,

expression levels of these genes were low in both NIL-

hbd2/hbd2 ? hbd3/hbd3 and Habataki CKI1 over-expres-

ser compared to the pathogen infected plants (Mitsuhara

et al. 2008), thus indicating that the immune response

signal in this hybrid breakdown is activated at a lower level

than that of an actual pathogen attack.

Molecular mechanism of hybrid breakdown

Results of this study suggest that the most likely mecha-

nism involved in hybrid breakdown is the autoimmune

response activated by one or more NBS-LRR genes in the

hbd3 locus. NBS-LRRs are involved in an ‘‘effector-trig-

gered immunity’’ system in which NBS-LRR recognizes

avirulence (Avr) proteins (Chisholm et al. 2006; DeYoung

and Innes 2006). Avr proteins are specialized pathogen

effectors that confer virulence function in the absence of

the cognate R gene. NBS-LRR can recognize Avr proteins

either directly (Jia et al. 2000; Dodds et al. 2003) or indi-

rectly (Mackey et al. 2002; Axtell and Staskawicz 2003;

Coaker et al. 2005; Mucyn et al. 2006). In the indirect

recognition mechanism, NBS-LRR detects changes in the

inner cellular component of the cells brought about by Avr

proteins.

In Drosophila and yeast, CKI1 families are involved in

numerous cellular events such as the cell cycle, morpho-

genesis, circadian rhythm, and signal transduction (Gross

and Anderson 1998; Knippschild et al. 2005). Through the

analysis of antisense lines, the CKI1 in this study is

reportedly involved in root development and hormone

sensitivity (Liu et al. 2003). However, there was no evi-

dence suggesting that it is involved in the immune

response.

Thus, the most plausible and probable mechanism

model in this hybrid breakdown is that hbd2-CKI1 acts like

the Avr protein or a protein disturbed by the Avr protein,

with one or more NBS-LRRs in hbd3 recognizing hbd2-

CKI1 directly or indirectly to trigger the immune response

signal.

Restricted distribution of hbd2 in rice accessions

Our analysis of the distribution of the hbd2 mutation

among 69 accessions in the World Rice Collection (Kojima

et al. 2005) revealed that this mutation is present only in

Milyang 23, a modern indica variety (Fig. 5). Further

analysis revealed that hbd2 is derived from Peta, an indica

type Indonesian landrace (Fig. 5). Thus, hbd2 currently has

less impact in rice speciation. The mutation may be lost or

fixed in some population through neutral drift, positive

selection with an unexpected favoring effect or genetic

hitchhiking. Thus, this hybrid breakdown is only at the

initial step of BDM incompatibility establishment.

Mol Genet Genomics (2010) 283:305–315 313

123

Evolutionary force in the establishment of BDM

incompatibilities in plants

The importance of the autoimmune response in hybrid

weakness as amply discussed by Bomblies and Weigel

(2007) is supported by the results of this study. In addition,

we also suggest that NBS-LRRs are important contributors

to hybrid weakness, as have been reported in some recent

studies (Bomblies et al. 2007; Alcazar et al. 2009). NBS-

LRR constitutes a large family in plants than in other

organisms, numbering about 500 in rice (Yang et al. 2006)

and about 150 in Arabidopsis (Meyers et al. 2003).

In addition, NBS-LRRs tend to diversify due to diversify-

ing selection (Mondragon-Palomino et al. 2002; Yang et al.

2006). These conditions imply that plant NBS-LRRs are

very likely to recognize more substances beside the Avr

protein, including gene products produced in other popu-

lations or species. We predict that trivial mutations, such as

the hbd2 mutation, have the potential to become a target of

highly diversified NBS-LRR(s) from other individuals or

species. Thus, plant NBS-LRRs have a greater potential to

establish hybrid weakness and to become an evolutionary

force in plant speciation compared to other gene classes.

Hybrid weakness has been reported not only in rice

(Amemiya and Akamine 1963; Sato and Morishima 1988;

Fukuoka et al. 1998, 2005, Kubo and Yoshimura 2002;

Matsubara et al. 2007; Yamamoto et al. 2007; Miura et al.

2008), but also in other plant species (Bomblies and

Weigel 2007). Identifying the causal genes of this phe-

nomenon in other plants will determine the validity of this

hypothesis.

Acknowledgments We thank Mr. Naoya Watanabe and Dr. Yasu-

hiro Kondoh (Honda Research Institute, Japan) for helpful sugges-

tions regarding the experimental design and Ikuko Aichi and Midori

Ito for technical assistance. This study was supported by a Grant-in-

Aid from the Ministry of Education, Culture, Sports, Science, and

Technology, Japan (19688002 to M.A.) and research fellowships from

the Japan Society for the Promotion of Science for Young Scientists

(to E.Y.).

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