ORIGINAL ARTICLE

Analysis of nucleotide diversity among alleles of the majorbacterial blight resistance gene Xa27 in cultivars of rice(Oryza sativa) and its wild relatives

Waikhom Bimolata • Anirudh Kumar • Raman Meenakshi Sundaram •

Gouri Shankar Laha • Insaf Ahmed Qureshi • Gajjala Ashok Reddy •

Irfan Ahmad Ghazi

Received: 29 January 2013 / Accepted: 23 April 2013 / Published online: 8 May 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract Xa27 is one of the important R-genes, effective

against bacterial blight disease of rice caused by Xantho-

monas oryzae pv. oryzae (Xoo). Using natural population

of Oryza, we analyzed the sequence variation in the

functionally important domains of Xa27 across the Oryza

species. DNA sequences of Xa27 alleles from 27 rice

accessions revealed higher nucleotide diversity among the

reported R-genes of rice. Sequence polymorphism analysis

revealed synonymous and non-synonymous mutations in

addition to a number of InDels in non-coding regions of the

gene. High sequence variation was observed in the

promoter region including the 50UTR with ‘p’ value

0.00916 and ‘hw’ = 0.01785. Comparative analysis of

the identified Xa27 alleles with that of IRBB27 and

IR24 indicated the operation of both positive selection

(Ka/Ks [ 1) and neutral selection (Ka/Ks & 0). The

genetic distances of alleles of the gene from Oryza nivara

were nearer to IRBB27 as compared to IR24. We also

found the presence of conserved and null UPT (upregulated

by transcriptional activator) box in the isolated alleles.

Considerable amino acid polymorphism was localized in

the trans-membrane domain for which the functional sig-

nificance is yet to be elucidated. However, the absence of

functional UPT box in all the alleles except IRBB27 sug-

gests the maintenance of single resistant allele throughout

the natural population.

Keywords Allele mining � Allelic diversity � Bacterial

blight � Synonymous mutation � Nonsynonymous mutation �Trans-membrane domain

Abbreviations

BB Bacterial blight

NBS-LRR Nucleotide binding site leucine rich repeats

RLL Relative lesion length

TSS Transcription start site

UTR Untranslated region

UPT Upregulated by transcriptional activator

NJ Neighbor joining

Introduction

Plants are constantly subjected to attacks from various

biotic stresses such as different diseases. Both the plants

and the pathogens have been co-evolving in response to

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00425-013-1891-3) contains supplementarymaterial, which is available to authorized users.

W. Bimolata � A. Kumar � I. A. Ghazi (&)

Department of Plant Sciences, School of Life Sciences,

University of Hyderabad, Prof. C. R. Rao Road,

Gachibowli, Hyderabad 500046, India

e-mail: irfan@uohyd.ernet.in

W. Bimolata

e-mail: bimowai@gmail.com

R. M. Sundaram � G. A. Reddy

Crop Improvement Section, Directorate of Rice Research,

Rajendranagar, Hyderabad 500030, India

e-mail: rms@drricar.org

G. S. Laha

Crop Protection Section, Directorate of Rice Research,

Rajendranagar, Hyderabad 500030, India

e-mail: gslaha@drricar.org

I. A. Qureshi

Department of Biotechnology, School of Life Sciences,

University of Hyderabad, Prof. C. R. Rao Road,

Gachibowli, Hyderabad 500046, India

e-mail: iaqsl@uohyd.ernet.in

123

Planta (2013) 238:293–305

DOI 10.1007/s00425-013-1891-3

each other’s modulation. Resistance (R)-genes play a major

role in the first line of defense against pathogens. R-genes

interact with their cognate avirulent (avr) genes encoded by

the pathogens and mediate host plant resistance. This

interaction between the host and its pathogen is believed to

play a major role in evolution of genetic variations in the

R-genes of plants and virulence/avirulence genes of

pathogens. The loss of R-gene mediated resistance is

attributed to mutation in effector gene leading to genetic

changes during the course of evolution which alter the

effector’s structure and function. An in-depth analysis of

molecular evolution and maintenance mechanism of

resistance and susceptibility alleles of R-gene may unravel

the role of pathogen-imposed selection of R-genes (Tiffin

and Moeller 2006).

In rice, the molecular evolution and genetic diversity of

genes related to disease response have been widely studied

for the major blast resistance gene, Pi-ta (Huang et al.

2008; Yoshida et al. 2009; Lee et al. 2011). Other R-genes

which have been extensively studied in the context of

sequence diversity are ‘Pm3’ locus in wheat conferring

resistance against powdery mildew (Bhullar et al. 2009),

Rpm1 (Stahl et al. 1999), RPS2 (Caicedo et al. 1999) and

RPP13 (Rose et al. 2004) in Arabidopsis, Cf2 in tomato

(Caicedo et al. 2004) and RGC2 in lettuce (Kuang et al.

2004). Similar studies have been done for disease response

genes in higher plants such as loblolly pine (Ersoz et al.

2010); however, till date, genetic diversity analysis of any

of the rice bacterial blight (BB) resistance genes has not

been reported. Bacterial blight caused by different races of

the bacterium Xanthomonas oryzae pv. oryzae (Xoo) is one

of the oldest known and ravaging diseases of rice. This

disease hampers rice production in Asia up to 60 % (Re-

issig 1986) and is predominantly rampant in humid tropical

and temperate regions. Till date, more than 30 BB resis-

tance genes have been identified and six of these have been

cloned and functionally characterized (Chun et al. 2012).

The existence of natural variation for a particular gene

provides the platform to identify multiple allelic variations

contributing different phenotypic patterns of resistance

(Iyer-Pascuzzi et al. 2007).

Xa27, a major BB R-gene identified from Oryza minuta

(BBCC), is one of the six BB resistance genes, which have

been cloned. Its function and mechanism of action has been

well characterized (Gu et al. 2005, 2009; Romer et al.

2009). It is a dosage-dependent, differentially expressed

R-gene which confers resistance against Xoo PXO99A. It

also shows broad-spectrum resistance against diverse

strains of Xoo obtained from different Asian countries

including two Indian strains A3842 and A3857 (Gu et al.

2004). Plants with Xa27 gene show resistance only against

incompatible strains of Xoo, harboring the avirulence gene

avrXa27. Xa27 is an intronless gene encoding a protein of

113 amino acids (Gu et al. 2005). Its recessive allele xa27

from IR24 also codes for the same protein without any

change in the protein sequence. The main polymorphic

regions between these two alleles are localized upstream of

transcription start site (TSS) and TATA box. There is a

deletion of three nucleotides ‘AGA’ at -51 position in the

promoter of the recessive allele of the gene (i.e., xa27, Gu

et al. 2005). Unlike other well-known R-genes, Xa27 does

not code any known conserved domains like NBS-LRR or

LRR receptor kinase and has been predicted to encode a

protein with a trans-membrane domain (Gu et al. 2005).

With the availability of Xa27 sequence in public domain,

allelic diversity analysis of wild relatives and landraces

may help in unlocking new and wide spectrum resistant

alleles. Analysis of DNA polymorphism for Xa27 in Indian

accessions of two major wild species of rice, O. nivara and

O. sativa may offer a prospect to divulge the process of

fixation of alleles and haplotypes responsible for adaptive

variation during the course of evolution. It will also reveal

the level to which the genes and other regulatory systems

are conserved across the species (Koornneef et al. 2004).

In the present study, we analyzed inter and intraspecific

polymorphism in Xa27 locus of Asian cultivated rice O.

sativa and its ancestor O. nivara from 27 different wild rice

accessions, introgression lines and landraces. The major

objectives of this study were: (1) to determine if the Xa27

alleles of O. sativa and its wild relative share identity; (2)

to analyze the level of polymorphism of Xa27 at the

sequence level and explain its molecular evolution; and (3)

to analyze the possibility of selection at Xa27 locus.

Materials and methods

Plant materials and growth conditions

A total of 27 accessions of different Oryza species were

analyzed for sequence diversity of Xa27 locus (Online

Resource 1). IR24, PB1 and TN1 were also used as disease-

sensitive control. The germplasms were obtained from

Directorate of Rice Research, Hyderabad and NBPGR,

New Delhi (India). Rice seeds were germinated in dark at

28 �C in germination box layered with pre-soaked germi-

nation paper. After a week, the germinated seeds were

shifted to 10-cm diameter pots containing soil. The seed

beds were uniformly watered and fertilized with a half-

strength Hoagland nutrient solution. After 30 days, healthy

seedlings were transplanted to 20-l earthen pots. Five pots

were kept for each genotype with three plants in each pot

filled with a mixture of clay and peat (1:1, v/v). The wild

accessions were propagated as tillers.

294 Planta (2013) 238:293–305

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Bacterial strains, plant inoculation and disease

phenotyping

After 45 days of transplantation, each accession was

challenged with five Indian virulent isolates of Xoo. The

isolates used for screening were DX011 (Pantnagar, Utta-

rakhand), DX127 (Cuttack, Orissa), DX020 (Hyderabad,

Andhra Pradesh), DX015 (Aduthurai, Tamil Nadu) and

DX133 (Raipur, Chhattisgarh). These strains were obtained

from Department of Plant Pathology, DRR, Hyderabad,

and cultured on modified Wakimoto’s medium at 28 �C for

72 h. The bacteria were then scraped and suspended in

sterile distilled water and the concentration was adjusted to

0.1–0.2 OD (1 9 108–1 9 109 CFU/ml). Using this bac-

terial suspension, 5–6 uppermost leaves of plants at the

booting stage were inoculated following leaf clipping

method (Kauffman et al. 1973). Three plants were used for

each isolate. The control plants were clipped with scissors

dipped in sterile water. After 15 days of inoculation, lesion

lengths were measured and the ratios of lesion length to

leaf length (RLL) were calculated. Scoring of disease was

done following standard evaluation system for rice (SES

scale; IRRI 2002). RLLs\25 % were scored as resistant or

tolerant genotypes and [25 % were scored as susceptible.

During the process of screening for bacterial blight resis-

tance, seedlings were uniformly irrigated and fertilized

using Hoagland nutrient solution.

DNA isolation, gene amplification and sequencing

Genomic DNA was isolated from young leaves following

CTAB method (Doyle and Doyle 1990) with minor mod-

ifications (Rajendrakumar et al. 2007). The entire Xa27

gene including the promoter and 30UTRs were amplified

from 27 genotypes using overlapping gene-specific primers

designed from GenBank accession AY986492 (Fig. 1).

Genotypes which were either highly resistant or moder-

ately resistant to most of the strains were selected along

with few susceptible ones. Whole gene amplification was

performed using high-fidelity jump start AccuTaq poly-

merase. PCR was carried out in 30 lL reaction volume

containing 50 ng of genomic DNA, 0.3 lM of each primer,

0.2 mM of each dNTPs, 109 PCR buffer (10 mM Tris–

HCl, pH 8.0, 2.5 mM MgCl2) and 0.75 U of AccuTaq

DNA polymerase (Sigma-Aldrich). The PCR profile con-

sisting of 3 min initial denaturation at 96 �C, 30 cycles of

amplification with 30 s DNA denaturation at 94 �C, 45 s

annealing at 60 �C and 2 min 15 s elongation at 68 �C was

adopted. Subsequently, the amplified products were visu-

alized on 1 % agarose gels. The PCR products were then

purified using gel extraction kit (Sigma-Aldrich), cloned

into pTZ57R/T (Fermentas) and sequenced. The sequenced

alleles were deposited in NCBI GenBank.

Allelic diversity and sequence data analysis

Based on the sequences obtained from the genotypes under

study, the coding regions and the UTR regions of the new

alleles were predicted through the software utility, FGE-

NESH (http://www.softberry.com) (Salamov and Solovyer

2000). Xa27 CDS of IRBB27 (GenBank accession

AY986492) and its recessive allele from IR24 (GenBank

accession AY986491) were used as reference for compar-

ative analysis. The percentage identity of each alleles and

their deduced protein sequence with that of Xa27 (IRBB27)

and xa27 (IR24) were determined by pairwise alignment.

Pairwise alignment was performed in BLASTN and

BLASTP (Altschul et al. 1990), respectively. Intraspecific

and interspecific polymorphisms were analyzed using

DnaSP program version 5.0 (Librado and Rozas 2009).

Sequences were assembled and subjected to multiple

alignments using ClustalX version 1.81 (Thompson et al.

1997) and Multalin version 5.4.1 (Corpet 1988). The

aligned file was used as an input for analysis in DnaSP

(http://www.ub.edu/dnasp). The numbers of polymorphic

sites, including SNPs, InDel polymorphism, synonymous

and non-synonymous substitutions in coding and non-

coding region (Nei and Gojobori 1986) were determined

according to DnaSP. All the analysis was done in sliding

window for 50 bp per window. Alignment gaps were

excluded from comparative analysis. Nucleotide diversity

was analyzed by estimating p (average number of nucle-

otide diversity per site Nei and Li 1979); and hw, (number

of segregating sites, Watterson 1975). Initially, each allele

was compared to Xa27 (IRBB27) and xa27 (IR24),

respectively. Later on, overall comparative analysis for all

the sequenced alleles was performed. Different test selec-

tion analysis such as Tajima’s D test (Tajima 1989) and Fu

Fig. 1 A schematic presentation of Xa27 gene. Overlapping primers were designed covering the entire gene. The arrows represent the regions

and the orientations of the primers designed

Planta (2013) 238:293–305 295

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and Li’s D test (Fu and Li 1993) were conducted separately

for O. nivara and O. sativa accessions using DnaSP pro-

gram to determine departures from neutrality. A dendro-

gram predicting the evolutionary relationship among 26

alleles was generated by developing bootstrapped unrooted

linear neighbour joining (NJ) tree in MEGA 4 (Tamura

et al. 2007). We also used maximum parsimony and

maximum likelihood methods to construct the tree; how-

ever, there was not much difference found from NJ plot.

The robustness of the tree was determined by taking

bootstrap value up to 100,000. To analyze the level of

diversity in regulatory region, the alleles were divided into

three parts; promoter with 50UTR, 30UTR and coding

region. Sequence diversity analysis was also done sepa-

rately for O. nivara and O. sativa accessions. The presence

of trans-membrane helices in the coded protein were pre-

dicted using TOPCONS (Bernsel et al. 2009) and signal

peptide prediction was done with the help of SignalP 4.0

(Petersen et al. 2011). SIFT analysis was done for the

deduced protein sequence to determine the deleterious

nature of the mutation (Kumar et al. 2009).

Results

Phenotypic evaluation

Out of the 27 accessions selected for allelic diversity

analysis, 10 O. nivara accessions were resistant to all the

five strains and three of them were susceptible to either one

of the strain; both O. alta and O. officinalis were resistant

to all the five strains, whereas among the O. sativa acces-

sions TN1, IR24 and IR20 were susceptible to all the five

Xoo strains and remaining accessions were resistant or

susceptible to one or the other strain (Online resource 1).

Gene amplification and sequencing

The sequence encompassing the entire Xa27 gene was

amplified from 27 rice accessions using overlapping gene-

specific primers. Sequencing was done for all the cloned

alleles. The size of the sequenced alleles ranged from 2,056

to 2,733 bp in length. Overall sequence similarity of the

entire gene ranged from 95 to 99 %. The deduced proteins

of Xa27 alleles from WR132, WR11, WR14, WR82,

WR110 and ON15 (O. nivara); PAU933 and R9 (O. sativa)

coded for 112 amino acids while the remaining alleles

encoded proteins of 113 amino acids, with few differences

in the protein sequence (Tables 1, 2). In WR14, WR82,

WR110, ON15, PAU933, R9 and WR132, three basepairs

(TAC) deletion (Online Resource 2) was observed in the

coding region which lead to deletion of threonine

(T) (Fig. 2a). However, deletion of nucleotide ‘G’ in

WR11 at 90th position (Online Resource 2) resulted in a

frameshift mutation as shown in Fig. 2a. All the sequences

were deposited in NCBI GenBank database and the

following accession numbers JF304301–JF304303,

HQ888852–HQ888857, JN016505–JN016521 and JN601064

were obtained.

Polymorphism of Xa27 alleles

The alleles from 29 genotypes including IR24 and IRBB27

were aligned. The total alignment length ranged from 2,056

to 2,780 bp, including sites with alignment gaps, promoter,

50 and 30UTR. Comparative analysis in DnaSP, excluding

sites with gaps, showed 103 polymorphic sites (in total

1,999 available sites), 74 singleton variable sites, 40 par-

simony informative site and 32 InDel events ranging from

1 to 50 bp in length. The average number of nucleotide

difference, ‘K’ of the entire gene was estimated to be

15.892. The genetic diversity, ‘p’ of the tested gene was

0.00795 and ‘hw’ equal to 0.01312 (Table 3). Among all

accessions, Mustilaiphou was found to be highly diverse

from IRBB27 (p = 0.02291) than IR24 (p = 0.0163)

(Tables 4, 5). The highest sequence variability was

observed in the 50 flanking region of the alleles with ‘p’

value 0.00916 and least variability in 30UTR. The test of

neutrality failed to give significant Tajima’s and Fu and

Li’s D (P [ 0.01) in O. nivara and O. sativa species, even

though hw value was greater than p resulting in negative

Tajima’s D (Table 3). InDel polymorphism was also

detected predominantly in promoter region with few cases

in exonic part. Some of the InDel sites were consistent

between the species. Fixed differences of nucleotide pat-

tern were detected among O. sativa and O. nivara alleles

with few cases of variation within the species too. Intra-

specific polymorphism was lower in O. nivara

(p = 0.00790) than O. sativa (p = 0.00845) (Table 3).

Deletion of &400 bp from the upstream region of Xa27

alleles was noticed in WR132, WR56, WR82, PAU933,

ON15, and WR110. Details of InDel polymorphisms and

SNPs detected in comparison to IR24 and IRBB27 was

summarized in Tables 1 and 2. The level of amino acid

polymorphism was highest in O. nivara. Nine alleles from

species O. nivara had single amino acid replacement,

whereas WR56 (two changes), WR1 and ON44 (three

changes) had more than one. Single amino acid replace-

ment was seen only in three accessions of O. sativa

(Fig. 2a). Both conservative and non-conservative amino

acid polymorphisms were present in the alleles, out of

which, two conservative and three non-conservative amino

acid polymorphism lie in the first trans-membrane domain;

however, these mutations did not make any major changes

in the trans-membrane structure. SIFT analysis also

showed that the amino acid mutations in the alleles were

296 Planta (2013) 238:293–305

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tolerable. In WR11, TOPCONS prediction showed the

absence of trans-membrane (TM) region and N-terminal

signal peptide, whereas in the remaining alleles, presence

of TM was predicted at two regions from 32 to 52 residues

and 69–89 residues (Fig. 2b, c).

The promoter region of the alleles other than IRBB27

shared similar UPT box to that of IR24 with deletion of

three nucleotides ‘AGA’ at -51 position (from transcrip-

tion start site) and substitutions from ‘C’ to ‘A’ (Fig. 3)

which is crucial for binding of AvrXa27 that in turn acti-

vates the transcription of the gene (Romer et al. 2009).

These three nucleotides were proved to be part of the UPT

(upregulated by transcription activator) box, a stretch of 16

nucleotides just after the TATA box which actually binds

the AvrXa27 protein (Romer et al. 2009; Bogdanove et al.

2010).

Xa27 allele tree and molecular evolution

An unrooted NJ plot based on the nucleotide variations in

Xa27 locus was constructed to analyze the phylogenetic

relationships among the alleles. An unrooted tree has no

evolutionary direction since the history of evolution and

origin of these alleles cannot be traced solely from the

available sequence data. The entire coding and non-coding

part of the sequences were considered to generate the tree.

When all the 29 sequences were considered for construct-

ing the tree, due to low polymorphism among O. sativa

accessions, a monophyletic clade was formed and was

difficult to get a resolved tree with higher bootstrap value.

Maximum likelihood and maximum parsimony also gen-

erated similar pattern of tree. Hence, three nearly identical

sequences of O. sativa were removed and only 26

Table 1 Summary of sequence comparison in the entire region of Xa27 among Oryza species using IRBB27 (resistant) as reference

Species Accession Genome Entire region (bp) Exon region (bp) 50UTR (bp) 30UTR (bp) Protein (a.a.)

Total Identity

(%)

Total SNPs Ia Total SNPs Ia Total SNPs Ia Total Similarity

(%)

O. sativa IRBB27 AA 2,361 – 342 – – 1,557 – – 462 – – 113 –

O. nivara WR01 AA 2,369 97 342 3 0 1,565 23 7 462 0 0 113 97

WR10 AA 2,405 97 342 1 0 1,601 12 5 462 0 0 113 99

WR11 AA 2,371 96 339 0 1 1,568 32 10 462 0 0 112 70

WR14 AA 2,387 97 339 7 1 1,586 26 9 462 0 0 112 95

WR56 AA 2,060 96 342 5 0 1,256 20 8 462 1 0 113 98

WR82 AA 2,056 96 339 8 1 1,255 16 9 462 1 0 112 95

WR85 AA 2,390 98 342 1 0 1,586 19 5 462 0 0 113 99

WR86 AA 2,390 97 342 1 0 1,586 24 5 462 1 0 113 100

WR102 AA 2,406 97 342 2 0 1,602 13 6 462 1 0 113 99

WR110 AA 2,057 98 339 7 1 1,256 15 8 462 0 0 112 95

ON15 AA 2,058 96 339 8 1 1,257 14 9 462 1 0 112 95

ON24 AA 2,341 97 342 2 0 1,537 36 7 462 1 0 113 99

ON34 AA 2,380 97 342 1 0 1,585 14 4 462 1 0 113 100

ON44 AA 2,383 97 342 3 0 1,579 25 7 462 0 0 113 97

WR132 AA 2,057 97 339 8 1 1,256 17 10 462 1 0 112 95

O. alta NB16 CCDD 2,406 97 342 2 0 1,602 13 4 462 1 0 113 94

O. officinalis NB40 CC 2,393 98 342 0 0 1,589 16 6 462 0 0 113 99

O. sativa PAU201 AA 2,394 97 342 1 0 1,590 11 7 462 1 0 113 100

PAU933 AA 2,058 96 339 5 1 1,257 23 9 462 0 0 112 95

R2 AA 2,392 97 342 0 0 1,588 15 6 462 0 0 113 100

R4 AA 2,407 97 342 1 0 1,603 13 5 462 1 0 113 100

R9 AA 2,364 96 339 5 1 1,563 34 15 462 0 0 112 95

R23 AA 2,393 98 342 1 0 1,589 11 11 462 0 0 113 100

IR20 AA 2,393 98 342 0 0 1,589 10 6 462 0 0 113 100

Mohem Phou AA 2,395 97 342 1 0 1,591 18 6 462 1 0 113 100

Musti Laiphou AA 2,733 99 342 1 0 1,929 52 20 462 1 0 113 100

TNI AA 2,395 97 342 1 0 1,591 11 6 462 0 0 113 99

a Number of InDel events

SNPsingle nucleotide polymorphisms, Data of 50UTR include promoter region

Planta (2013) 238:293–305 297

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sequences were considered for constructing the NJ plot.

Finally, we obtained a resolved asymmetric tree of higher

bootstrap value. Musti laiphou (ML) was shown as an out

group. From the phylogenetic tree, we observed two major

groups of sequence type where ML was the lone member in

the second group (Fig. 4). This observation may be the

result of insertion of &350 bp in the upstream region of

ML. The separation shown were mainly at the species level

as O. sativa and O. nivara formed separate clusters with the

exception of one or two accessions; however, the alleles

were not clustered on the basis of resistance/susceptible

genotype. The nucleotide as well as the protein sequence

similarity of WR1 (His-17, Phe-33, Val-40), ON24 (His-

17) and ON44 (His-17, Phe-33, Val-40) found in the same

clade suggests their similar origin. It was also observed that

all the susceptible alleles from IR24, IR20 and TN1, etc.,

belonged to same group. Furthermore, Xa27 gene in

IRBB27 which was originally derived from O. minuta was

found nearer to O. nivara than IR24 (O. sativa) from

evolutionary point of view. The resistant Xa27 allele and

those of other wild accessions might had evolved around

the same period.

Selection at the Xa27 locus

The Ka/Ks ratio was calculated for each allele with resis-

tant (IRBB27) and susceptible (IR24) allele to study their

divergence rate from the resistant and susceptible one.

Comparison of each allele individually with IRBB27 and

IR24 had shown that Ka/Ks was zero for 10 accessions,

namely IR20, NB40, WR11, WR86, Musti laiphou, Mo-

hem phou, R2, R4, PAU201 and ON34 (Tables 4, 5) which

Table 2 Summary of sequence comparison in the entire region of Xa27 gene among Oryza species using IR24 (susceptible) as reference

Species Accession Entire region (bp) Exon region (bp) 50UTR(bp) 30UTR (bp) Protein (a.a.)

Total Identity (%) Total SNPs Ia Total SNPs Ia T o tal SNPs Ia Total Similarity (%)

O. sativa IR24 2,393 – 342 – – 1,589 – – 462 – – 113 –

O. nivara WR01 2,369 97 342 3 0 1,565 17 5 462 0 0 113 97

WR10 2,405 99 342 1 0 1,601 4 4 462 0 0 113 99

WR11 2,371 95 342 0 1 1,564 28 11 462 0 0 113 70

WR14 2,387 97 339 7 1 1,586 31 11 462 0 0 112 95

WR56 2,060 97 342 5 0 1,256 15 10 462 1 0 113 98

WR82 2,056 97 339 8 1 1,255 11 10 462 1 0 112 95

WR85 2,390 97 342 1 0 1,586 24 7 462 0 0 113 99

WR86 2,390 97 342 1 0 1,586 30 6 462 1 0 113 100

WR102 2,406 99 342 2 0 1,602 5 4 462 1 0 113 99

WR110 2,057 98 339 7 1 1,256 10 10 462 0 0 112 95

ON15 2,058 97 339 8 1 1,257 9 9 462 1 0 112 95

ON24 2,341 95 342 2 0 1,537 35 7 462 1 0 113 99

ON34 2,389 97 342 1 0 1,585 10 6 462 1 0 113 100

ON44 2,383 97 342 3 0 1,579 21 9 462 0 0 113 97

WR132 2,057 98 339 8 1 1,256 12 11 462 1 0 112 94

O. alta NB16 2,406 99 342 2 0 1,602 5 1 462 1 0 113 99

O. officinalis NB40 2,393 98 342 0 0 1,589 6 0 462 0 0 113 99

O. sativa PAU201 2,394 99 342 1 0 1,590 1 1 462 1 0 113 100

PAU933 2,058 96 339 5 1 1,257 18 11 462 0 0 112 95

R2 2,392 99 342 0 0 1,588 0 0 462 0 0 113 100

R4 2,407 99 342 1 0 1,603 5 3 462 1 0 113 100

R9 2,364 97 339 5 1 1,563 28 10 462 0 0 112 95

R23 2,393 99 342 1 0 1,589 1 0 462 0 0 113 100

IR20 2,393 100 342 0 0 1,589 0 0 462 0 0 113 100

Mohem Phou 2,395 99 342 1 0 1,591 7 6 462 1 0 113 100

Musti Laiphou 2,733 99 342 1 0 1,929 37 16 462 1 0 113 100

Taichung Native 1 2,395 98 342 1 0 1,591 1 1 462 0 0 113 99

a Number of InDel events

SNP single nucleotide polymorphisms

298 Planta (2013) 238:293–305

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means only synonymous changes were present. It suggests

that the selection was neutral for these accessions. Even

though we found lot of sequence diversity in and around

the promoter and 50UTR of Musti laiphou, non-synony-

mous change was not detected. High rate of amino acid

replacement was also observed in 50 % of alleles where

Fig. 2 a Output of multiple alignment for the predicted amino acid

sequences of Xa27 alleles. The rice genotypes are indicated in left

column. Alignment was performed using Multalin program. The

numbers on the top of the sequences indicate the position of amino

acids. Insertions and deletions are shown with gaps. Different colors

in amino acid shows non-synonymous changes. Frameshift mutation

in WR11 is also shown in the bottom. TOPCONS prediction for

presence of trans-membrane (TM) domain in IRBB27 (b) and WR11

(c). Depression in peaks at two regions around 32–52 residues and

69–89 residue (X-axis) in IRBB27 shows the presence of two TM

domain whereas it is absent in WR11. Position of predicted TM is

indicated by white and grey bar

Table 3 Polymorphism and neutral test of different regions of the Xa27 gene

Total sites (excluding sites with gaps) S p hw Fu and Li’s D Tajima’s D K

Entire gene 1,999 103 0.00795 0.01312 – – 15.892

Coding 338 17 0.01202 0.01281 – – 4.064

Promoter and 50UTR 1,198 84 0.00916 0.01785 – – 10.975

O. nivara seq 2,004 57 0.00790 0.00875 -0.82225 -0.41969* 15.829

O. sativa seq 2,039 73 0.00845 0.01186 -1.48507 -1.6476* 17.22

* P [ 0.01 (not significant), S no. of polymorphic sites, K average nucleotide difference, p nucleotide diversity, hw no. of segregating sites

Planta (2013) 238:293–305 299

123

rate of non-synonymous changes by rate of synonymous

changes (Ka/Ks) was [1. Alleles of TN1 and R23 when

compared to IR24 showed very high Ka/Ks & 8.00, where

the rate of amino acid replacement was occurring at a faster

rate than the synonymous changes which suggest rapid

adaptive evolution (Bergelson et al. 2001). However, when

compared with IRBB27, the rate of amino acid changes

was substantially lower with Ka/Ks = 0.788. Along with

positive selection, we could also see purifying selection in

some of the alleles, ON24, WR85, NB16, WR102 and

WR56 where Ka/Ks \ 1 (Tables 4, 5).

Discussion

Natural variation in the alleles of R-genes due to SNPs,

InDels and insertion or deletions of large DNA fragments

plays an important role in providing phenotypic diversity

or variability with respect to disease resistance. Consid-

ering the existence and emergence of virulent pathotypes

of the bacterial blight pathogen, there is a need to exploit

the potential of allele mining by identification of novel

alleles of R-genes. Study on molecular evolution and

understanding genetic diversity of known R-gene(s) will

also help us in finding key region(s) controlling disease

resistance. Maintenance of allelic diversity at R-gene in

natural population occurs as a result of co-evolution of

host and pathogen (May and Anderson 1983). Pathogens

are believed to play an important role as a selective agent

during the course of R-gene evolution (Kover and Schaal

2002). In the present study, we initially identified a set of

rice genotypes possessing resistance against multiple

virulent strains of Xoo and analyzed the nucleotide

and amino acid sequence diversity with respect to Xa27,

an R-gene involved in gene-for-gene interaction with

AvrXa27.

Table 4 Summary of nucleotide diversity, total number of sites, average number of nucleotide difference, synonymous and non-synonymous

sites, rate of non-synonymous substitution by synonymous substitution with reference to IRBB27

Accession/code Total no. of site

(excluding gaps)

S p Synonymous (and non-coding) sites Non synonymous sites Ka/Ks

No. of sites No. of mutation No. of sites No. of mutation

ON34 2,357 16 0.00679 2,111.67 16 245.33 0 0.00

NB16 2,358 16 0.00679 2,112.67 15 245.33 1 0.5774

PAU201 2,356 13 0.00552 2,110.67 13 245.33 0 0.00

R4 2,358 15 0.00636 2,112.67 15 245.33 0 0.00

R2 2,356 15 0.00637 2,110.67 15 245.33 0 0.00

Mohem Phou 2,356 20 0.00849 2,109.67 20 245.33 0 0.00

Musti Laiphou 2,357 54 0.02291 2,111.67 54 245.33 0 0.00

WR102 2,357 16 0.00679 2,111.33 15 245.67 1 0.5774

WR86 2,358 26 0.01103 2,112.67 26 245.33 0 0.00

WR10 2,357 13 0.00552 2,111.33 12 245.57 1 0.7192

WR11 2,340 32 0.01368 2,095.33 32 242.67 0 0.00

WR132 2,032 25 0.01230 1,788.17 20 240.83 5 1.8672

ON24 2,336 39 0.01670 2,090.33 38 245.67 1 0.2228

WR110 2,033 22 0.01082 1,789.17 17 240.83 5 2.1979

WR85 2,358 20 0.00848 2,113.00 19 245.00 1 0.4555

R9 2,339 39 0.01667 2,095.17 34 240.83 5 1.2865

PAU933 2,033 28 0.01377 1,789.17 23 240.83 5 1.6230

R23 2,356 12 0.00509 2,110.67 11 245.33 1 0.7884

WR82 2,032 25 0.01230 1,788.17 20 240.83 5 1.8672

WR56 2,036 26 0.01277 1,791.33 24 244.67 2 0.6074

WR1 2,354 26 0.01105 2,108.50 23 245.50 3 1.1181

ON44 2,356 28 0.01188 2,110.50 25 245.50 3 1.0336

WR14 2,353 33 0.01402 2,109.17 28 240.83 5 1.5746

ON15 2,033 23 0.01131 1,789.17 18 240.83 5 2.0891

NB40 2,356 16 0.00679 2,110.00 16 245.33 0 0.00

IR20 2,356 10 0.00424 2,110.67 10 245.33 0 0.00

TN1 2,356 12 0.00509 2,110.67 11 245.33 1 0.7886

S no. of polymorphic sites, p nucleotide diversity, Ka/Ks ratio of rate of amino acid replacement over rate of synonymous changes

300 Planta (2013) 238:293–305

123

Unique SNPs were present in the 50UTR of NB40,

WR14, Musti laiphou, PAU933, PB1, WR11, R9 and

nearby region of UPT boxes in Mohem phou, WR11, R9,

WR1, ON24, PAU933, Pusa Basmati1, R4 and WR102.

Similarly, identical polymorphisms were found in wild rice

accessions ON44, ON24 and WR1 (Fig. 3) both in the

protein coding region as well as the UPT box region and

these accessions were clustered in the same clade at the

phylogenetic tree. At the species level, we observed a set of

SNPs and InDels unique to O. nivara and O. sativa,

respectively, in the genomic region encompassing Xa27 but

some polymorphic traits were common in both. Intraspe-

cific polymorphism was less in both O. sativa and O.

nivara which may be due to small sample size. Moderate

interspecific polymorphism was detected in the promoter

region of Xa27 locus. A lesser amino acid polymorphism at

the intraspecific level may be due to purifying selection

resulting in conservation of amino acid sequence as

reported in the case of R-gene loci RPS5, RPM1, RPS2 and

RPP13 in Arabidopsis thaliana (Caicedo et al. 1999;

Mauricio et al. 2003). Low level of polymorphism was also

reported for other R-genes in rice such as most widely

studied Pita conferring resistance against M. grisea (Huang

et al. 2008; Yoshida and Miyashita 2009; Lee et al. 2011).

At the genome level, NB16 (CCDD genome), NB40 (CC)

and IRBB27 did not show much sequence divergence from

the AA genome (O. sativa and O. nivara); earlier studies

have also shown close relatedness and colinearity between

AA and CC genome (Vaughan 1994; Zou et al. 2008; Feng

et al. 2009). Highly conserved colinearity between AA and

CCDD genome was also reported by Huang et al. (1994).

Xa27 alleles showed an average polymorphism of

ps = 0.00795. This low variation is comparable with that

of O. sativa, which has a nucleotide diversity (psilent) in the

range of 0.0011–0.0035 (Tang et al. 2006; Zhu et al. 2007)

and also ‘Pita’ locus of O. rufipogon with p = 0.00235

Table 5 Summary of nucleotide diversity, total number of sites, average number of nucleotide difference, synonymous and non-synonymous

sites, rate of non-synonymous substitution by synonymous substitution with reference to IR24

Accession/code Total no. of site

(excluding gaps)

S p Synonymous (and non-coding) sites Non-synonymous sites Ka/Ks

No. of sites No. of mutation No. of sites No. of mutation

ON34 2,365 12 0.00507 2,119.67 12 245.33 0 0.00

NB16 2,391 8 0.00335 2,145.67 7 245.33 1 1.24

PAU201 2,393 3 0.00125 2,147.67 3 245.33 0 0.00

R4 2,392 7 0.00293 2,146.67 7 245.33 0 0.00

R2 2,392 0 0.00 2,146.67 0 245.33 0 0.00

Mohem Phou 2,391 9 0.00376 2,147.67 9 243.33 0 0.00

Musti Laiphou 2,393 39 0.0163 2,147.67 39 245.33 0 0.00

WR102 2,391 8 0.00335 2,145.33 7 245.67 1 1.24

WR86 2,375 32 0.01347 2,129.67 32 245.33 0 0.00

WR10 2,390 5 0.00209 2,144.33 4 245.67 1 2.15

WR11 2,348 28 0.01193 2,103.33 28 242.67 0 0.00

WR132 2,040 21 0.01029 1,796.17 16 240.83 5 2.344

ON24 2,336 38 0.01627 2,090.33 37 245.67 1 0.2290

WR110 2,041 17 0.00833 1,797.17 12 240.83 5 3.14

WR85 2,374 25 0.01053 2,129.00 24 245.00 1 0.3596

R9 2,352 33 0.01403 2,108.17 28 240.83 5 1.5746

PAU933 2,041 23 0.01127 1,797.17 18 240.83 5 2.0891

R23 2,393 2 0.00084 2,147.67 1 245.33 1 8.20

WR82 2,040 20 0.00980 1,796.17 15 240.83 5 2.5119

WR56 2,044 21 0.01027 1,799.33 19 244.67 2 0.7735

WR1 2,365 20 0.00846 2,119.50 17 245.50 3 1.5185

ON44 2,363 24 0.01016 2,117.50 21 245.50 3 1.2300

WR14 2,369 38 0.01604 2,125.17 33 240.83 5 1.3439

ON15 2,042 18 0.00881 1,798.17 13 240.83 5 2.8904

NB40 2,393 6 0.00251 2,147.67 6 245.33 0 0.00

IR20 2,393 0 0.00 2,147.67 0 245.3 0 0.00

TN1 2,393 2 0.00084 2,147.67 1 245.33 1 8.00

S number of polymorphic sites, p nucleotide diversity, Ka/Ks ratio of rate of amino acid replacement over rate of synonymous changes

Planta (2013) 238:293–305 301

123

(Huang et al. 2008). Nucleotide divergence for O. nivara

accessions also were much lower with ‘p’ = 0.0079

(Table 3). Yoshida and Miyashita (2009) also reported

very low diversity at silent site of ‘Pita’, ps = 0.0101.

However, we may get higher allelic variability if we

include more number of species such as O. rufipogon, O.

longistaminata or O. minuta, etc., in the study. We may

also conduct this study in different sets of Oryza species for

better understanding of Xa27 molecular evolution. The

nucleotide polymorphism in 50 non-coding region

(p = 0.00916) was shown to be much higher than that of

the entire region (Table 3). This may be due to the low

polymorphisms of the 30UTR and exclusion of sites with

gaps from the analysis. We found that the genetic diversity

of Xa27 alleles was in and around the range of those

reported defense-related genes. Nucleotide diversity of

other defense-related genes such as Adh3 locus in wild

barley has p value 0.0219 (Lin et al. 2001); Adh loci of

allogamous species depicted p values from 0.00204 to

0.01742 (Cummings and Clegg 1998). In A. thaliana, a

chitinase-encoding gene has p = 0.0104 (Kawabe et al.

1997) and Pto, disease resistance gene of wild tomato

(Lycopersicon) was reported with p = 0.012 (Rose et al.

2007). Bakker et al. (2008) also reported that seven genes

encoding pathogen-related proteins, had ps = 0.00183 and

pa = 0.00126.

The divergence rate ratios for most of the O. nivara

accessions from IRBB27 were high with Ka/Ks [ 1 which

suggest the occurrence of positive selection or adaptive

evolution (Bergelson et al. 2001). Most of the O. sativa

accessions exhibited neutral selection with Ka/Ks = 0,

while few O. nivara and ILs have Ka/Ks \ 1 illustrating

the presence of selection constraint. Even though, WR11

(O. nivara) showed zero divergence rate from IRBB27, at

the translation level, it encoded a non-functional protein

due to frame shift mutation. This miscalculation arose due

to the exclusion of single base pair deletion from the

analysis by the software. We also observed similar pattern

of divergence rate between the susceptible allele xa27

(IR24) and the new alleles. The rate of diversity was very

much higher than those observed between Xa27 (IRBB27)

and the alleles. A strong positive selection was shown in

TN1, R23 and WR110 against IR24. This observation was

due to very low polymorphism at silent sites (ps) of the said

alleles. In general, adaptive evolution is most commonly

observed in regions involved in host pathogen recognition

(Endo et al. 1996) such as in the case of Xa21 and Xa21D,

occurrence of non-synonymous mutation was higher than

synonymous mutation in LRR domain (Wang et al. 1998).

For thorough analysis of adaptive and positive selection of

R-genes, we should consider only those sites predicted to

play an important role in recognition (Wang et al. 1998).

Amino acids evolve at a faster rate in functionally impor-

tant regions of R-proteins than the corresponding rate of

synonymous changes (Meyers et al. 1998). Similar pattern

was observed in this case with majority of the amino acid

replacement taking place in the TM domain despite the fact

that this substitution did not cause major changes in the

pattern of the TM. Probably, Xa27 was evolving under

relaxed constraint. The role of these TMs in pathogen

recognition is yet to be explored in order to understand the

resistance mechanism of Xa27 completely.

Fig. 3 Multiple alignment of the promoter region of Xa27 covering

the predicted UPTAvrXa27. The rice genotypes are indicated in left

column. Multiple alignment was constructed in Multalin. Deletion of

three bp ‘AGA’ and substitution of ‘C’ by ‘A’ in the UPT box is

indicated by black bar. It is at -51 position of the gene (from TSS)

302 Planta (2013) 238:293–305

123

When Tajima’s D was calculated for O. nivara acces-

sions and O. sativa accessions, the difference between pand h was very less which failed to give a significant

D value. The non-significant Tajima’s D and Fu and Li’s

F may be due to small sample size and random sampling.

Simultaneously, we cannot overrule the possibility of

selection occurring at this locus: firstly, as the samples

under study were selected randomly from a natural popu-

lation and not from an interbreeding population; secondly,

Xa27 locus interacts directly with the pathogen where the

possibility of co-evolution of both plant and pathogen exist.

So, a population-level study of R-gene locus will yield

accurate measurement of selection (Caicedo et al. 1999).

Other than looking only at the coding region for selection,

we found high level of nucleotide polymorphism at the

promoter and 50UTR among the accessions which pos-

sessed many regulatory sites. The roles of these polymor-

phisms in the gene function are yet to be explored and this

region may also be under natural selection. More impor-

tantly, the deletion and substitution in the UPTAvrXa27 box

in all alleles except IRBB27 will make these alleles unable

to signal AvrXa27 recognition (Romer et al. 2009), which

suggest the selective maintenance of dominant Xa27 or we

can say the gain of function of this allele during the course

of evolution among the natural population. This might also

be the possible case of an ‘‘evolutionary arms race’’ or a

negative frequency-dependent selection. A more detailed

study with more number of natural populations may pro-

vide crucial insights to understand the exact evolutionary

dynamics of Xa27 locus.

Twenty-seven alleles of Xa27 showing numerous

sequence diversity have been identified. Other than the few

changes in the coded protein sequences, the UPT box was

similar to that of IR24. We presume that the binding

affinity of AvrXa27 to the UPT box of these alleles will be

weak due to presence of defective UPT box and hence

binding of AvrXa27 will be unable to induce the expres-

sion of the alleles. It may be interesting to study the dif-

ferent alleles where amino acid changes had occurred, after

fusing these newly found coding sequences with a func-

tional UPTAvrxa27 by promoter engineering as described by

Romer et al. (2009) and Hummel et al. (2012). Clear

understanding of functional difference among alleles will

help in deciphering its structure and function (Bergelson

et al. 2001) as well as studying the molecular diversity of

avrxa27 will clear the co-evolutionary process of this R-avr

gene. The available sequence data of different alleles and

the level of diversity found at the Xa27 locus may provide

great opportunity for studying the evolution of plant–

pathogen interactions as well as a chance to study both

evolutionary and genetic aspects of this unique R-gene.

Acknowledgments The authors are grateful to two anonymous

reviewers for their critical review and valuable suggestions to

improve the manuscript. The research is supported by Department of

Science and Technology, New Delhi, India, Grant no. SR/SO/PS-21/

09. The authors also thank DRR, Hyderabad, Rajendra Agricultural

College, Pusa (Bihar) and NBPGR, New Delhi, for providing the

germplasms. Financial supports from XI plan seed money, DBT-

CREBB, DST-FIST, UGC-SAP-CAS-I and maintenance grant from

University of Hyderabad is gratefully acknowledged. W.B. is also

grateful to CSIR for the fellowship (Award no. 09/414/(0832)/2008-

EMR-I).

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