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Euphytica (2008) 160:411422

DOI 10.1007/s10681-007-9564-6

Marker assisted introgression of bacterial blight resistance


in Samba Mahsuri, an elite indica rice variety
Raman M. Sundaram Manne R. Vishnupriya
Sunil K. Biradar Gouri S. Laha Gajjala Ashok Reddy
N. Shobha Rani Nukala P. Sarma
Ramesh Venkata Sonti

Received: 24 February 2007 / Accepted: 28 August 2007 / Published online: 18 September 2007
 Springer Science+Business Media B.V. 2007

Abstract Samba Mahsuri (BPT5204) is a medium


slender grain indica rice variety that is very popular
with farmers and consumers across India because of
its high yield and excellent cooking quality. However, the variety is susceptible to several diseases and
pests, including bacterial blight (BB). We have used
PCR based molecular markers in a backcross-breeding
program to introgress three major BB resistance
genes (Xa21, xa13 and xa5) into Samba Mahsuri
from a donor line (SS1113) in which all the three
genes are present in a homozygous condition. At each
backcross generation, markers closely linked to the
three genes were used to select plants possessing
these resistance genes (foreground selection) and
microsatellite markers polymorphic between donor
and recurrent parent were used to select plants that

Electronic supplementary material The online version of


this article (doi:10.1007/s10681-007-9564-6) contains supplementary material, which is available to authorized users.
R. M. Sundaram and M. R. Vishnupriya have contributed
equally to this work.
R. M. Sundaram  S. K. Biradar  G. S. Laha 
G. A. Reddy  N. S. Rani  N. P. Sarma
Directorate of Rice Research, Rajendranagar, Hyderabad
500030, India
M. R. Vishnupriya  R. V. Sonti (&)
Centre for Cellular and Molecular Biology, Uppal Road,
Hyderabad 500007, India
e-mail: sonti@ccmb.res.in; rvsonti@yahoo.com

have maximum contribution from the recurrent


parent genome (background selection). A selected
BC4F1 plant was selfed to generate homozygous
BC4F2 plants with different combinations of BB
resistance genes. The three-gene pyramid and twogene pyramid lines exhibited high levels of resistance
against the BB pathogen. Under conditions of BB
infection, the three-gene pyramid lines exhibited a
significant yield advantage over Samba Mahsuri.
Most importantly, these lines retain the excellent
grain and cooking qualities of Samba Mahsuri
without compromising the yield as determined in
multi-location trials. This work demonstrates the
successful application of marker-assisted selection
for targeted introgression of multiple resistance genes
into a premium quality rice variety.
Keywords Bacterial blight  Gene pyramiding 
Marker assisted selection  Resistance genes 
Rice

Introduction
Rice (Oryza sativa L.) is an important food crop that
serves as a major carbohydrate source for nearly half
of the worlds population. Although a large number
of rice varieties have been released for different agroecosystems in India, only a few are widely grown.
Rice variety BPT5204, popularly known as Samba
Mahsuri, is one of the rice varieties, which is very

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412

widely grown in India. It was developed by A.N.G.R.


Agricultural University in the state of Andhra
Pradesh (Reddi et al. 1979). This variety is extremely
popular amongst rice farmers and consumers because
of its high yield, medium slender grains and excellent
cooking and eating qualities. Although the variety
was initially released for cultivation in Andhra
Pradesh in Southern India, it has now spread to five
other states occupying *3.3% of the rice growing
area in the country (Directorate of Rice Research
2006). This amounts to more than one million
hectares and this figure is expected to go up
significantly in the near future.
Despite its popularity, Samba Mahsuri is highly
susceptible to many pests and diseases including
bacterial blight (BB). This disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), is the most serious
bacterial disease of rice and has become a major
production constraint after the wide-spread cultivation of plant type based high yielding nitrogen
responsive rice varieties. In India, the extent of yield
losses can be as high as 50% (Rao and Kauffman
1971). BB is particularly severe in the irrigated and
rainfed lowland ecosystems (Mew 1987) in which
Samba Mahsuri is cultivated.
Chemical control for BB is not effective (Devadath
1989). Therefore, host plant resistance offers the most
effective, economical and environmentally safe
option for management of BB (Khush et al. 1989).
Globally, more than twenty-five BB resistance genes
have been identified from diverse sources (Chu et al.
2006a). A number of these resistance genes have been
tagged by closely linked molecular markers (Sonti
1998; Rao et al. 2002). A few of these genes like Xa4
have been incorporated widely in many high yielding
varieties through conventional breeding (Khush et al.
1989). However, widespread cultivation of varieties
with Xa4 has led to predominance of Xoo races that
can overcome this gene (Mew et al. 1992). The
deployment of rice cultivars that have multiple BB
resistance genes is expected to lead to more durable
resistance.
DNA fingerprinting and pathotype analysis have
indicated that there is a significant amount of
diversity within populations of Xoo in India and
other rice growing countries (Adhikari et al. 1995;
Yashitola et al. 1997; Shanti et al. 2001; Gupta et al.
2001; Singh et al. 2003). A number of near-isogenic
rice lines in the genetic background of IR24 were

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Euphytica (2008) 160:411422

inoculated with Indian strains of Xoo and those


containing the single resistance genes Xa21, xa13 and
xa5 were found to provide moderate to strong levels
of resistance (Goel et al. 1998; Shanti et al. 2001;
Joseph et al. 2004; our unpublished results). PCR
based molecular markers that are tightly linked to
each of these three resistance genes have been
developed (Ronald et al. 1992; Yoshimura et al.
1995; Zhang et al. 1996) and used to pyramid them
into the genetic background of different rice varieties
(Huang et al. 1997; Sanchez et al. 2000; Singh et al.
2001; Joseph et al. 2004). The three-gene pyramids
containing Xa21, xa13 and xa5, developed through
these studies, have been tested against multiple
Indian isolates of Xoo and were found to exhibit
high levels of resistance.
Considering the importance of Samba Mahsuri to
the Indian rice economy, and the necessity to
introduce BB resistance, we have used marker
assisted backcross breeding to introgress the Xa21,
xa13 and xa5 resistance genes into this variety.
Marker assisted selection is particularly useful for
this purpose because xa13 and xa5 are recessive
resistance genes. In the absence of markers, identifying backcross plants that have these genes would
require progeny testing, which is an addition of one
more generation and cumbersome too. Also, none of
the Indian Xoo strains in our collection are compatible with either Xa21 or xa13. Therefore we would
not be able to distinguish, through phenotypic
screens, rice lines that have only Xa21 or xa13 from
those having both the genes. Because of these
reasons, marker-assisted selection was considered to
be necessary for introgression of these resistance
genes into Samba Mahsuri. A number of mapped rice
microsatellite markers (*500) were available when
this program was initiated in April 2000 (Panaud
et al. 1996). Microsatellite markers are highly
sequence specific and co-dominant. These features
make them ideal for use in background selection as
they permit the detection of alleles coming from both
parents and allow accurate determination of the
allelic constitution of the offspring. Hence, we used
a set of microsatellite markers that are polymorphic
between the donor and recurrent parents in selection
for the recurrent parent genome at each backcross
generation.
The newly developed lines that were obtained in
this study exhibited high BB resistance and retain the

Euphytica (2008) 160:411422

yield and grain quality traits of Samba Mahsuri. This


work represents a successful example of the use of
molecular markers, in foreground and background
selection, for introgression of genes of interest into a
premium rice variety.

Materials and methods


Plant materials
The donor line was a near isogenic line in the genetic
background of PR 106, named SS1113, possessing
three BB resistance genes, Xa21, xa13 and xa5 (Singh
et al. 2001). The recurrent parent was a BB susceptible cultivar, Samba Mahsuri (also called BPT 5204),
which is an elite medium slender grained rice variety
released by A. N. G. R. Agricultural University,
Hyderabad, India. In addition, the Taichung Native 1
(TN 1) rice variety was used as a BB susceptible
check.

Marker assisted selection


Mini-scale DNA isolation for PCR analysis of the
parents and backcross progenies for foreground
selection was carried out from 25-day old seedlings
following the procedure of Zheng et al. (1995).
Three STS markers, pTA248, RG136 and RG556,
closely linked to the BB resistance genes, Xa21,
xa13 and xa5, respectively (Huang et al. 1997),
were used to confirm the presence of the resistance
allele of each gene at each backcross generation.
The pTA248 marker is *0.2 cM from Xa21 (Ronald
et al. 1992), the RG136 marker is *1.5 cM
from xa13 (Zhang et al. 1996; our unpublished
data) and the RG556 marker is *0.1 cM from xa5
(Yoshimura et al. 1995). The PCR mixture contained 50 ng template DNA, 5 pmoles of each
primer, 0.05 mM dNTPs, 1 PCR buffer (10 mM
Tris, pH 8.4, 50 mM KCl, 1.8 mM MgCl2 and
0.01 mg/ml gelatin) and 1 U of Taq DNA polymerase in a reaction volume of 25 ll. Template DNA
was initially denatured at 94C for 5 min followed
by 35 cycles of PCR amplification with the following parameters: a 30-s denaturation at 94C, a 30-s
annealing at 55C and 12 min of primer extension
(1 min for pTA248, 2 min for RG136 and RG556)

413

at 72C. A final extension was done at 72C for


7 min. The amplified product of pTA248 was
electrophoretically resolved on a 1.2% agarose gel
containing 0.5 lg/ml of ethidium bromide in 0.5
TBE buffer and visualized under UV. For the
amplified products of RG136 and RG556, 5 ll of
PCR product was used for gel electrophoresis to
determine whether PCR was successful. The remaining PCR product was used for restriction digestion.
The reaction mixture consisted of 0.6 ll (10 U/ll)
of restriction enzyme (Hinf1 for RG136 amplicons
and Dra1 for RG556 amplicons), 2.5 ll of 10
restriction buffer, 2.4 ll of sterile distilled water and
20 ll PCR product. The reaction mixture was
incubated for 4 h at 37C and the products of
restriction digestion were separated by gel electrophoresis (1.5% agarose) and visualized under UV
after staining with ethidium bromide.
For background selection, genomic DNA was
isolated from 60-day old plants as per the procedure
of Dellaporta et al. (1983). Microsatellite loci that
are polymorphic between donor and recurrent parents were identified by screening 139 microsatellite
markers distributed throughout the rice genome
(Table 1). PCR was carried out using 5 ng DNA
as template for amplification, 5 pmoles of each
primer, 0.05 mM dNTPs, 1 PCR buffer (Perkin
Elmer, Wellesley, MA), 100 lM dUTP (Flourescein) and 1 U Taq DNA polymerase in a total volume
of 12.5 ll. Template DNA was initially denatured
for 94C for 5 min followed by 35 cycles of PCR
amplification with the following cycling conditions
of 30 sec denaturation at 94C, 30 sec primer
annealing at 55C (or as described in Chen et al.
1997 and Panaud et al. 1996), 1 min extension at
72C and a final extension of 72C for 5 min. PCR
products along with TAMRA 500 internal size
standard (Applied Biosystems Inc [ABI], Foster
City, CA), were separated on 5% denaturing PAGE
using an ABI PRISM 377 automatic sequencer and
analyzed with Genescan software as per the manufacturers instructions. In this screen, 50 markers
were identified to be polymorphic between SS1113
and Samba Mahsuri. The parental polymorphic
microsatellite markers were used to genotype foreground selected plants at each backcross generation
to estimate the amount of recurrent parent genome
contribution G which was calculated as per the
following formula:

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414
Table 1 Microsatellite
markers that are
polymorphic between
SS1113 and Samba Mahsuri

Genomic DNA was


isolated, PCR was
performed and
polymorphisms analyzed
using primers for 139
different rice microsatellite
markers as described in
Materials and Methods

The number of
polymorphic markers/
chromosome and their
RM#s are indicated in
parentheses

Euphytica (2008) 160:411422

Chromosome locus

Total # of markers
analyseda

# of Polymorphic
markersb

16

7 (RM 220, 243, 23, 81, 24, 237, 246)

14

4 (RM 154, 324, 240, 207)

3
4

13
10

1 (RM 16)
3 (RM 335, 317, 280)

10

3 (RM 153, 249, 13)

11

2 (RM 162, 276)

13

8 (RM 125, 214, 320, 336, 10, 70, 18, 248)

12

8 (RM 337, 25, 310, 72, 44, 210, 149, 230)

11

5 (RM 219, 257, 278, 245, 205)

10

10

3 (RM 239, 311, 271)

11

11

12

Total # of markers

139


G = [(X + 1/2Y)  100] N
where
N = total number of parental polymorphic markers screened
X = number of markers showing homozygosity for
recurrent parent allele
Y = number of markers showing heterozygosity
for parental alleles

2 (RM 21, 286)


4 (RM 19, 313, 235, 17)
50

content) for a number of physico-chemical characteristics by following standard procedures (Little


et al. 1958; Beachel and Stansel 1963; Juliano 1971;
Anonymous 2002). The characteristics that were
evaluated are milling (%), head rice recovery (%),
length breadth ratio, grain type, grain chalkiness,
alkali spreading value and amylose content.
Field evaluation of pyramided lines of Samba
Mahsuri

Assessment of BB resistance
The pyramided lines were evaluated for their reaction
to BB under glass house conditions using seven
isolates of Xoo obtained from different geographical
locations in India. Individual lines were grown in
plastic pots (17 cm diameter 15 cm height) under
flooded conditions with a mixture of soil and farmyard
manure (3:1 ratio). The pots were fertilized with N: P
@ 100:50 kg/ha with P applied as basal dose and N
applied both as basal dose and top dressing at 25 DAS
(days after sowing). Forty-day old plants were clip
inoculated (Kaufmann et al. 1973) with bacterial
suspensions of 109 cfu/ml. Approximately 1520
leaves were inoculated/pot and lesion lengths were
evaluated 15 days after inoculation.
Evaluation of grain quality characteristics
The grains of the pyramided lines were evaluated
(three months after harvest at 1214% moisture

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Four of the promising three-gene pyramid lines at


BC4F6 generation, along with the parents, were
evaluated at 10 selected locations across India during
the wet season (JuneNovember) in 2005 for grain
yield and other agro-morphological parameters (flowering duration, panicles/m2 and plant stature) using
standard procedures under Advance Variety Trial
1-Near isogenic lines (AVT 1-NIL) of All India
Coordinated Rice Improvement Programme (AICRIP). The AICRIP is organized by the Directorate
of Rice Research, an Institute of the Indian Council of
Agricultural Research.
In order to assess the effect of BB on yield, each of
the three-gene pyramid lines along with Samba
Mahsuri were grown, following a standard package
of practices, in 10 m2 plots in the experimental farm
of the Directorate of Rice Research during the wet
season of 2006. All plants in a plot were clip
inoculated with Xoo at the maximum tillering stage.
Lesion lengths were measured fifteen days after

Euphytica (2008) 160:411422

inoculation. Yield data was recorded at harvest from


inoculated and adjacent uninoculated plots. The
experiment was conducted in three replications.

Results
Introduction of BB resistance genes into Samba
Mahsuri background
The SS1113 rice variety (donor of the BB resistance
genes Xa21, xa13 and xa5) was crossed to Samba
Mahsuri with the former as the male parent. The F1
plants were confirmed for their heterozygosity for the
R gene linked markers and were backcrossed using
Samba Mahsuri as a female parent. The resulting
BC1F1 lines were first checked for presence of the
marker linked to Xa21 resistance allele in a heterozygous condition. All of the plants that were

Fig. 1 Foreground selection at BC1F1 generation using R


Gene linked PCR based markers. (A) Screening of plants using
Xa21 linked marker pTA248. (B) Screening of plants positive
for Xa21 using xa13 linked marker. (C) Screening of plants
positive for both Xa21 and xa13 using xa5 linked marker

415

heterozygous for Xa21 were checked for presence


of the marker linked to the xa13 resistance allele in a
heterozygous condition. Finally, the double positive
plants were screened for presence of the marker
linked to the xa5 resistance allele in a heterozygous
condition (Fig. 1).
A total of 11/145 BC1F1 plants were found to be
triple heterozygotes for the R gene linked markers.
These 11 plants were subjected to background
selection. Out of a total of 139 rice microsatellite
markers that were screened, 50 were found to be
polymorphic between the parents (Table 1). The
polymorphic markers were used to genotype the
eleven plants and identify the plant having maximum
recurrent parent genome contribution as described in
Materials and Methods. The selected plant having the
maximum recurrent parent genome contribution was
backcrossed to generate BC2F1 plants (Samba
Mahsuri as the female parent) and the process was

RG556. Lanes MMolecular weight Maker (Lambda EcoRI +


HindIII double digest), DDonor line (SS1113), R
Recipient line (Samba Mahsuri), 1 to 145BC1F1 plants.
Arrow indicates a positive plant

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Euphytica (2008) 160:411422

gene linked markers to identify plants that were


homozygous for different R genes or their combinations. As expected, plants that are homozygous for
all three R genes, lines with various two gene
combinations as well as single R gene containing
lines were obtained (Table 3).
Fig. 2 Background selection using a parental polymorphic
microsatellite marker. Genomic DNA was isolated from
SS1113, Samba Mahsuri and BC3F1 plants that are triple
heterozygotes for R gene linked markers. PCR was done using
RM153 marker and genotyping was performed as described in
Materials and Methods. The amplicons of SS1113 and Samba
Mahsuri are in lanes 1 & 2. Lanes 310 represent PCR products
obtained from eight BC3F1 lines. Lanes 4, 7 and 8 represent
plants that have become homozygous for the recurrent parent
allele. Lanes 3, 5, 6, 9 and 10 represent plants that are
heterozygous for the parental alleles

continued up to the BC4F1 stage. A representative


example of genotyping for background selection is
provided in Fig. 2. The number of triple heterozygotes amongst the backcross plants and the maximum
amount of estimated recurrent parent genome contribution to the selected triple heterozygote plant at each
backcross generation is provided in Table 2. It is to
be noted that, for as yet unknown reasons, the
contribution of the recurrent parent to the genome of
the backcross progeny was less than expected at each
of the backcross generations. At the BC4F1 generation, the plant having maximum contribution from the
recurrent parent (*96%; Table 2) was selfed to
obtain BC4F2 lines that were screened using the R

Evaluation of BB resistance characteristics


of pyramided lines
The pyramided lines were evaluated for their resistance to BB under glass house conditions using
several different isolates of Xoo. One of these isolates,
called BXO1, belongs to pathotype Ib, a widely
distributed Xoo pathotype in India (Yashitola et al.
1997). The lesion lengths obtained after inoculation
with BXO1 are shown in Fig. 3. As compared to
Samba Mahsuri, the leaves of the pyramided lines
(three gene and two gene combinations) exhibited
very small lesion lengths indicating a very high level
of resistance. In addition, the lines containing either
Xa21 alone or xa13 alone also exhibited limited lesion
lengths. However, extended lesions were observed on
lines containing only xa5. The three-gene pyramid
lines were inoculated with six other Xoo isolates from
different locations in India. One of these strains,
DX011, was isolated from Uttaranchal state in North
India and is a new pathotype that is different from Ib
(data not shown). Another strain, DX020, is a highly
virulent isolate of uncharacterized pathotype that was

Table 2 Number of triple R gene heterozygotes identified and estimation of recurrent parent genome contribution
# of plants
scoreda

# of plants that are


triple heterozygotesa,

BC1F1

145

BC2F1

126

BC3F1
BC4F1

Generation

Estimated maximum %
contribution of recurrent
parent genome to selected
backcross plantc

Expected % contribution
of recurrent parent genome
to selected backcross plantd

11

72

75

10

80

87.5

120

89

93.754

80

96

96.875

At each backcross generation, genomic DNA was isolated from derivative lines and genotyping was performed using primers that
are tightly linked to R genes as described in Materials and methods

At each backcross generation, fewer than expected triple heterozygotes were obtained. This is due to the fact that some of the
putative backcross progeny were obtained by inadvertent selfing of Samba Mahsuri (the female parent in these backcrosses)

At each backcross generation, genomic DNA was isolated from derivative lines that are triple heterozygotes for R gene linked
markers. Microsatellite markers that are polymorphic between the parental lines were then used, as described in Materials and
Methods, to identify the plant with maximum recurrent parent genome contribution

As per Mendelian ratios for independent gene action

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Euphytica (2008) 160:411422

417

Table 3 Number of lines with multiple R gene combinations


#

Gene combinations

SS1113 X BPT 5204 BC4Fa2

Line numbers

Xa21/Xa21 xa13/xa13 xa5/xa5

B-170, 189, 197, 210, 226

Xa21/Xa21 xa13/xa13 Xa5/Xa5

B-24, 29, 60, 69, 277

3
4

Xa21/Xa21 Xa13/Xa13 xa5/xa5


xa21/xa21 xa13/xa13 xa5/xa5

4
2

B-159, 199, 261, 266


B-48, 173

Xa21/Xa21 Xa13/Xa13 Xa5/Xa5

B-142, 249, 270, 276

xa21/xa21 xa13/xa13 Xa5/Xa5

B-9, 32, 182, 187

xa21/xa21 Xa13/Xa13 xa5/xa5

B-136, 184

A total of 320 BC4F2 plants were genotyped with R gene linked markers as described in Materials and Methods. As per
Mendelian ratios, the expected number of BC4F2 plants for each genotype listed above is 5 as these genes are independently inherited
(they are non-linked)

Fig. 3 Evaluation of bacterial blight resistance in gene


pyramid lines. Rice plants that were forty days old were
inoculated with saturated cultures of the BXO1 strain of
X. oryzae pv. oryzae as described in Materials and Methods.
Lesion lengths were measured 15 days after inoculation. Error
bars indicate the standard deviation of readings from ten
inoculated leaves. (1) Taichung Native-1 (susceptible check);

(2) Samba Mahsuri; 3 to 28 represent backcross progeny with


different R gene combinations; 3 to 7: Xa21 xa13 xa5; 8 to12:
Xa21 xa13; 13 to 16: Xa21 xa5; 17 to 18: xa13 xa5; 19 to 22:
Xa21; 23 to 26: xa13; 27 to 28: xa5. The results from one
experiment are presented and similar results were obtained
from three independent experiments

obtained from the experimental farm of the Directorate of Rice Research. The three-gene pyramid lines
were highly resistant to all six strains of Xoo with
average lesion lengths being \2 cm.

traits with Samba Mahsuri. Particularly, the pyramided lines showed a desirable alkali spreading value
of 5.0, which is identical to Samba Mahsuri and
different from SS1113 (7.0). In a panel test for taste
conducted by the grain quality-testing laboratory at
the Directorate of Rice Research, the pyramided lines
are indistinguishable from Samba Mahsuri.

Grain quality characteristics of pyramided lines


The donor line (SS1113) has long slender grains as
compared to Samba Mahsuri, which has medium
slender grains. Like Samba Mahsuri, the three-gene
pyramid lines have medium slender grains (Table 4).
Four of the three-gene pyramided lines (B170, B189,
B197, B226) were tested and found to be fully
comparable in milling, grain and cooking quality

Yield and agro-morphological traits of pyramided


lines
Four of the three-gene pyramid lines at BC4F6
generation {entries IET19046 (B226), IET19045
(B189), IET19026 (B197) and IET19590 (B170)}

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Euphytica (2008) 160:411422

Table 4 Grain quality parameters of the three-gene pyramid lines of Samba Mahsuria,b
Variety

Milling Head rice


Kernel length Kernel breadth L/B Grain Grain
%
recovery % (mm)
(mm)
ratio typec chalkiness

Alkali
Amylose
spreading content (%)
value

B197

71.5

68.1

5.00

1.73

2.89 MS

Absent

5.0

24.60

B189

70.5

66.5

4.86

1.83

2.66 MS

Absent

5.0

24.89

B226

69.7

65.6

5.06

1.84

2.75 MS

Absent

5.0

24.68

B170

24.39

71.3

67.8

4.87

1.94

2.51 MS

Absent

5.0

Samba Mahsuri 70.5

66.7

4.89

1.86

2.63 MS

Absent

5.0

24.21

SS1113

69.4

6.57

1.95

3.37 LS

Very occasionally 7.0


present

24.81

71.3

The grain quality parameters were determined as described in Materials and Methods

The experiments were performed twice with similar results

MS and LS indicate medium slender and long slender, respectively

along with the donor and recipient parents were


evaluated under an Advance Variety Trial of All
India Coordinated Rice Improvement Programme
(AICRIP) in the year 2005 at 10 selected locations
across India (Directorate of Rice Research 2006). The
recipient parent, Samba Mahsuri recorded an overall
mean grain yield of 4,739 kg/ha, while the donor
parent recorded 4,486 kg/ha. The test entries viz.,
IET Nos: 19046, 19045, 19590 and 19026 showed
grain yields on par with Samba Mahsuri at all the test
locations with marginal differences (Table 5). The
test entries did not show any significant variation as
compared to Samba Mahsuri in terms of flowering
duration, panicles/m2, plant stature as well as other
characters that are considered under distinctness,
uniformity and stability (DUS) tests. (Supplementary
Table 1; Directorate of Rice Research 2006).
The effect of BB on yield of Samba Mahsuri and
the three-gene pyramid lines (Table 6) was also
assessed by inoculation with Xoo strain DX020 as
described in Materials and Methods. Under BB free
conditions, there was no significant difference in
yield between Samba Mahsuri and the three-gene
pyramid lines. Under BB infection, there was a
*23% reduction in yield of Samba Mahsuri while
yield reduction was negligible for the three-gene
pyramid lines.

Discussion
Samba Mahsuri is a premium rice variety whose
popularity lies in its high yield and exceptional grain

123

and cooking quality. In the present work, we have


introgressed three BB resistance genes, Xa21 xa13
and xa5 into Samba Mahsuri with the objective of
developing BB resistant lines that retain the yield and
quality characteristics of Samba Mahsuri. The pyramided lines (containing either three R genes or two
R genes) exhibited a high level of resistance upon
inoculation with Xoo. The lines containing either of
the single resistance genes Xa21 or xa13 also
exhibited a good level of resistance. However, the
derivative lines containing only the xa5 resistance
allele exhibited a partial/marginal level of resistance.
This is consistent with our studies using the near
isogenic lines IRBB5 (containing xa5), IRBB13
(xa13) and IRBB21 (Xa21) which indicated that
xa13 and Xa21 provided a high level of resistance
while xa5 provided a moderate level of resistance
upon inoculation with the BXO1 strain of Xoo
(unpublished results). Although the lines containing
either Xa21 or xa13 alone or the various two-gene
pyramids are resistant to Xoo, we have performed
further characterization (with regard to yield and
quality parameters) only on the three-gene pyramid
lines as we expect that the resistance conferred by
them would be more durable and are therefore
targeting them for commercial release.
The lesion lengths that are presented in this paper
(Fig. 3) were obtained 15 days after inoculation.
When the inoculated leaves were examined 21 days
after inoculation, the lesions had expanded in the
single gene (Xa21 or xa13) containing lines and the
two-gene pyramid lines but not in the three-gene
pyramid lines (data not shown). This suggests that

Non-significant
d

Each entry into the All India Coordinated Rice Improvement Programme is given a unique IET (Initial Evaluation Trial) number

Grain yield of entries given in Kg/ha Standard error; values given are mean of three replications

The location average includes the yield data of the lines mentioned in the table as well as one local check line which varied depending on the location
c

50

0.9
18.2

NS
NS

7.5
19.6

NS
868

7.6
13.6

NS
672

12.9
2.0
5.4
CV%

208
440

NSd

4487

LSD (P \ 0.05)

8.7

3085

1896 21
3907 36

3732
4166

3981 26
5303 200

5284
6455

6931 504
3508 218

4229
2937

2043 179
6382 58

5709
6593

4159 55

4608
Location averagec

6766 563

4665

419

SS1113

4739

4574
3939 12

3628 15
4420 198

3504 59
3888 120

4444 41
4797 290

5050 333
6031 458

6111 351
3903 400

4250 281
2551 87

2825 138
5851 100

4546 33
6200 252

Samba Mahsuri

6465 495

4870 109

4945 182

IET19590 (B170)

4727

4667
3749 19

2696 38
3651 172

3432 479
4259 57

3611 115
6313 333

5050 333
5793 153

6560 318
4946 387

4946 263
2688 24

2784 33
5460 67

5319 100

4686 161
IET19046 (B226)

6561 273

4719 155
IET19045 (B189)

6713 231

4597
2723 23
3223 385
4259 88
5797 467
5846 88
4270 319
3155 77
5780 88
5294 72
IET19026 (B197)

6627 435

Mean
Maruteru
Hyderabad
Nawagam
Karjat
Raipur
Cuttack
Bhubaneswar
Kaul
Ludhiana
Pant Nagar
Varietyb

Table 5 Grain yield of three-gene pyramid lines along with donor and recipient lines as recorded in Advanced variety trial 1-NIL of All India Coordinated Rice Improvement
Programmea

Euphytica (2008) 160:411422

there is some kind of quantitative complementation


wherein the presence of multiple resistance genes has
an additive effect on the overall level of resistance.
Higher levels of resistance in gene pyramid lines
containing multiple BB resistance genes as compared
to lines having single (or fewer) resistance genes have
been reported earlier (Yoshimura et al. 1996; Huang
et al. 1997).
All the three resistance genes that have been
considered in the present work have been cloned and
characterized. Xa21 is a dominant resistance gene
that encodes a receptor kinase containing NBS-LRR
domains (Song et al. 1995), while xa5 is a recessive
resistance gene and encodes a variant form of
transcription factor cIIa (Iyer and McCouch 2004).
The xa13 resistance gene is also recessive in nature
and has been shown to be a mutation in the promoter
region of a gene that is a homolog of the nodulin
MtN3 (Chu et al. 2006b). In rice lines containing the
dominant (susceptibility) allele of the gene, the
expression of the nodulin homolog is up regulated
upon infection with Xoo. It appears that the increased
expression of this gene is necessary for Xoo to grow
on rice. This up regulation does not occur in rice lines
containing the resistance (recessive) xa13 allele
(Yang et al. 2006). The apparently different modes
of action of the three resistance genes used in this
work might contribute to make the resistance in the
three-gene pyramid lines quite durable.
At the time of initiation of this project, the number
of rice microsatellite markers that were available in
the public domain were *500. A much larger number
(*20,000) of rice microsatellite markers are currently
available. By screening through this new set of
markers, we have identified a total of 122 additional
microsatellite markers that are polymorphic between
SS1113 and Samba Mahsuri. These include 15, 9 and
9 markers respectively on chromosomes 3, 6 and 11
for which very few markers were used in the
background selection. We find that, in the three-gene
pyramids, 118 out of these 122 markers were homozygous for the Samba Mahsuri allele at BC4F6 (data
not shown). This indicates a recovery of *97% of the
Samba Mahsuri genome in the three-gene pyramid
lines, a number which is close to the value presented
in Table 2 with respect to recovery of recurrent parent
genome at BC4F1. Interestingly, 2 out of the 4 markers
that exhibited the SS1113 allele were tightly linked to
the xa5 and xa13 resistance loci.

123

420

Euphytica (2008) 160:411422

Table 6 Effect of bacterial blight (BB) on yield of Samba Mahsuri and three-gene pyramid lines
Line

Yield (kg/ha) under


BB free condition*

Yield (kg/ha) under


BB infection*

Lesion length
(cm)#

% of yield reduction
due to BB

Samba Mahsuri

6850 403

5250 296

24.66 2.87

23.36

B170

6613 451

6432 498

2.20 0.29

2.72

B189

7205 623

6913 447

2.20 0.57

4.05

B197

6932 455

6731 405

1.95 0.72

2.89

B226

6695 432

6525 393

2.21 0.32

2.54

*Experiments conducted in 10 m plots in three replications as described in Materials and Methods. Yield of 10 m2 plots extrapolated
to per ha yield (1 ha = 10,000 m2)
#Average lesion length for 25 leaves/plot measured 15 days after inoculation

The grain quality characteristics of the three-gene


pyramid lines are very similar to those of the parent
Samba Mahsuri line. In a panel test for taste, the
three-gene pyramid lines were indistinguishable from
Samba Mahsuri. Further, replicated field trials were
carried out at ten different locations across India
under the All India Coordinated Rice Improvement
Program. The yield levels of the three-gene pyramid
lines were not significantly different from those of
Samba Mahsuri indicating that there is no apparent
yield penalty associated with presence of the resistance genes. Under BB infection, there was a
significant yield reduction for Samba Mahsuri while
this was negligible for the three-gene pyramids. This
indicates that cultivation of the three-gene pyramid
lines would be of great advantage in BB endemic
areas.
The complete recovery of the yield and grain
quality characters of Samba Mahsuri in the threegene pyramid lines is a very significant achievement.
This is particularly so because yield and grain quality
are multigenic traits encoded by loci that are
distributed across the rice genome. Introgression of
resistance without recovery of yield and grain quality
characters would have been a futile exercise as the
developed lines would not be accepted by farmers
and Indian consumers who are extremely particular
about the grain and cooking quality. We feel that this
was made possible because of the extensive backcrossing (BC4) and the use of molecular markers for
background selection.
As per our analysis, we find that there is a
variation from the theoretically expected 75% contribution from the recurrent parent genome to the
BC1F1 plants. All the selected BC1F1 plants had a
recurrent parent genome contribution that was less

123

than the expected 75%. We have found similar results


for BC1F1 plants (data not shown) in another
backcrossing program for introgression of Xa21,
xa13 and xa5 from SS1113 into a rice cultivar called
Triguna. This suggests that selection for introgression
of the Xa21, xa13 and xa5 genes might be exercising
a pull, through as yet unknown mechanisms, which
favors inheritance of additional unlinked loci from
the donor genome in BC1F1 plants. In this study, a
less than expected contribution from the recurrent
parent was also observed in BC2F1 and BC3F1
generations. More experiments are needed to determine if such a pull does definitively exist.
However, it is clear from this work that background
selection with a limited number of polymorphic
microsatellite markers (*50), in conjunction with
four backcrosses is sufficient to recover the yield and
quality characteristics of the recurrent parent. Also, it
is pertinent to note that we have not performed
background selection with markers that are tightly
linked to (or flanking) the target traits. Background
selection with markers, which are closely linked to
the target traits is probably helpful, but not essential,
for recovery of recurrent parent characteristics following introgression of the Xa21, xa13 and xa5 genes
in rice.
As indicated above, these lines have undergone
one year of testing under the All India Coordinated
Rice Improvement Program. An additional year of
testing has been recently completed (data not shown)
and the results confirm the data presented in Table 5.
Subsequently, one of these lines (IET19046 [B226])
has been recommended for release as a commercial
variety by the variety identification committee of the
Indian Council of Agricultural Research (Directorate
of Rice Research 2007). In addition, these lines are

Euphytica (2008) 160:411422

being used as pre-breeding donor lines for transfer of


BB resistance in many rice breeding programs across
India. This work demonstrates that marker assisted
backcrossing can be gainfully employed for adding
new genes into popular and elite rice varieties that
have been grown by farmers over the years on
account of their unique characteristics. It can be
expected that the availability of the rice genome
sequence will facilitate the development of many
more markers for transfer of traits of agronomic
value, with greater precision, into commercially
important rice cultivars.
Acknowledgements We thank Drs. A. P. K. Reddy and
U. Prasada Rao for discussions. The encouragement and
support received from Drs. B. Mishra and B.C. Viraktamath
is thankfully acknowledged. The assistance received from
Mr. P. Natarajkumar and Mr. D. Surendher Reddy in
conducting the field experiments is also acknowledged. This
work was supported by a grant from the National Agricultural
Technology Project (NATP) of the Indian Council of
Agricultural Research. The authors would also like to thank
the scientists at the cooperating centers of AICRIP for
conducting the field trails.

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