Integrating marker assisted background analysis with foreground selection for
identification of superior bacterial blight resistant recombinants in Basmati rice
S. Gopalakrishnan1,2, R. K. Sharma3,4, K. Anand Rajkumar3, M. Joseph1, V. P. Singh1, A. K. Singh1,
K. V. Bhat5, N. K. Singh3and T. Mohapatra3,6
1Division of Genetics, Indian Agricultural Research Institute, New Delhi 110012;2National Bureau of Plant Genetic Resources
Regional Station, Jodhpur, Rajasthan;3National Research Centre on Plant Biotechnology, Indian Agricultural Research
Institute, New Delhi 110012;4Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh;5National Bureau
of Plant Genetic Resources, New Delhi 110012, India;6Corresponding author, E-mail: email@example.com
With 3 figures and 2 tables
Received August 1, 2006/Accepted September 3, 2007
Communicated by Z. K. Li
Basmati rice is highly susceptible to bacterial blight (BB) caused by
Xanthomonas oryzae pv. oryzae. Transfer of BB resistance genes from
non-Basmati sources to Basmati through cross-hybridization requires
strict monitoring for recovery of the desirable Basmati quality traits in
the recombinants, which show complex inheritance pattern. We
integrated background analysis using mapped microsatellite markers
with foreground selection to identify superior lines that combine useful
genes from a non-Basmati BB resistance donor line IRBB55 with grain
and cooking quality characteristics of the popular Basmati rice variety
?Pusa Basmati 1? (PB 1) employing backcross pedigree strategy.
Foreground selection using linked markers ensured presence of two
genes, xa13 and Xa21 for BB resistance from IRBB55, and the
recurrent parent PB 1 allele for the waxy locus giving intermediate
amylose content and maintainer allele at fertility restorer locus in the
BC1F5 recombinants. Background analysis enabled selection of
recombinants with recurrent parent genome to the extent of 86.3%
along with the quality traits. The extent of introgression of non-
Basmati donor chromosome segments in the superior selections was
estimated to be <7.8 Mb and <6.7 Mb in the xa13 and Xa21 linked
genomic regions, respectively. Association mapping identified three
quantitative trait loci, one each for 1000-grain weight, fertile grains/
panicle and cooked kernel length. The backcross-pedigree breeding
strategy facilitated recovery of additional desirable characteristics from
the donor in some of the selections. The elite selection Pusa 1460-01-
32-6-7-67 with maximum genomic background and quality character-
istics of the recurrent Basmati parent gave resistance reaction against
BB, similar to that of the non-Basmati resistant check variety and
recorded an yield advantage of 11.9% over the best check in the
multiplication agronomic trial in the Basmati growing region of India.
This line, which has been released as a new variety in the name of
?Improved Pusa Basmati 1? for commercial cultivation in India, is an
example of successful application of marker assisted selection to
Key words: Oryza sativa — Basmati rice — bacterial blight
resistance — amylose content — fertility restoration — marker
Basmati rice is unique for its harmonious combination of
pleasant aroma, fluffy texture of cooked rice, high volume
expansion during cooking made up by linear kernel elongation
with minimum breadth wise swelling, palatability, easy
digestibility and longer shelf life (Singh et al. 1988). High level
of susceptibility of Basmati rice to bacterial blight (BB), caused
by the bacterium Xanthomonas oryzae pv. oryzae (Xoo), is a
serious constraint to Basmati rice production. Besides, the
grain and cooking quality of the Basmati rice is severely
affected by this disease. There is no known source of BB
resistance in the available aromatic rice germplasm. Twenty-
eight major genes governing BB resistance have been reported
in the non-Basmati genotypes. As many as 12 of these genes
have been mapped using molecular markers (Ronald et al.
1992, Yoshimura et al. 1992a,b, 1995a,b), which are being
employed in marker assisted gene pyramiding for BB resis-
tance (Huang et al. 1997, Sanchez et al. 2000, Singh et al.
2001). Recently, we transferred BB resistance genes xa13 and
Xa21 from the non-Basmati donor line IRBB55 to a popular
high yielding Basmati cultivar ?Pusa Basmati 1? (PB 1) by
marker assisted foreground selection and identified recombi-
nants homozygous for both the genes in BC1F3generation
(Joseph et al. 2004). The economic worth of these recombi-
nants, however, depended upon the extent of recovery of
Basmati quality characteristics of the recipient parent.
Basmati quality traits are mostly quantitative in nature and
many of the loci affecting these traits have been mapped using
molecular markers. The linked markers, when validated across
different parental cross combinations, can be used in marker
assisted foreground selection in backcross breeding for recov-
ery of the quality traits. Among these traits, amylose content is
considered very important. The complex nature of inheritance
and complication in the measurement of amylose content
(Takeda et al. 1987) make it a difficult trait to improve in rice
breeding. The task is however, simplified due to the availability
of a stable microsatellite marker linked to waxy gene, a major
locus determining the amylose content in rice grains (Bligh
et al. 1995). Several studies have established strong association
between CTnrepeat number in waxy alleles and the amylose
content (Bergman et al. 2001, Larkin and Park 2003, Prathepa
and Baimai 2004). Use of this marker is expected to enhance
the efficiency of selection for amylose content.
Another approach that facilitates quick recovery of the
recipient parent genome including the desirable genic regions
employs marker assisted background selection (Hospital et al.
1992, Openshaw et al. 1994, Visscher 1996, Frisch et al.
1999a,b, Chen et al. 2000, 2001, Liu et al. 2003). Co-dominant
markers evenly distributed on all the chromosomes help
determine parental contributions to the progeny and design
graphic depiction of genotypes. This also allows reduction of
Plant Breeding 127, 131—139 (2008)
? 2008 The Authors
Journal compilation ? 2008 Blackwell Verlag, Berlin
linkage drag with reference to donor chromosome segment
linked to the gene(s) of interest (Brinkman and Frey 1977). Of
the various types of markers available for background
selection, the sequence tagged microsatellite site (STMS)
markers (Beckman and Soller 1990) mapped to specific
chromosomal locations that assay variation in simple sequence
repeat motifs are considered robust due to their co-dominance
and hyper-variability. In rice, high-density genome maps based
on STMS markers are available (Panaud et al. 1996, Cho et al.
2000, Temnykh et al. 2000, 2001, McCouch et al. 2002) that
can be effectively utilized in background analysis.
The present study was designed to identify desirable
recombinants that combine BB resistance with Basmati quality
traits in BC1F5generation derived from the cross between
Basmati variety ?Pusa Basmati 1? and non-Basmati donor of
BB resistance, IRBB55 developed earlier (Joseph et al. 2004).
Background analysis using mapped STMS markers was
integrated with foreground selection for BB resistance and
amylose content to identify superior lines with maximum
recovery of Basmati genome along with the quality traits and
minimum non-targeted genomic introgressions of the donor
chromosomes. Suitability of these lines as maintainers of male
sterility in breeding BB resistant cytoplasmic male sterile
(CMS) lines for development of heterotic Basmati rice hybrids
was explored using a STMS marker linked to the major
fertility restoration gene for the wild abortive (WA) cytoplasm-
based male sterility. The results of multilocation evaluation
over a period of 3 years leading to release of one of the elite
selections for commercial cultivation in India are reported.
Materials and Methods
Plant materials and screening for BB resistance: The plant materials of
rice, Oryza sativa L., used in the research work included (a) ?Pusa
Basmati 1? (PB 1), the most popular Basmati rice variety, which is
highly susceptible to bacterial blight (BB); (b) IRBB55, the donor
parent, carrying two BB resistance genes namely, xa13 and Xa21, in the
background of IR24, a widely grown non-Basmati rice variety
developed at the International Rice Research Institute, Philippines
and (c) thirty-one elite advanced generation lines (BC1F5generation)
derived by selfing the BC1F2individuals produced from the cross PB 1//
PB 1/IRBB55. The selections along with their parents were screened for
bacterial blight resistance in the field according to Joseph et al. (2004).
Molecular marker analysis: DNA isolation was carried out using the
standard Cetyl Trimethyl Ammonium Bromide method (Doyle and
Doyle 1990). Sequence tagged sites (STS) markers, RG136 (3.7 cM
away from xa13) and pTA248 (a gene sequence-based marker for
Xa21), were used to select for xa13 and Xa21 as given by Huang et al.
(1997). Marker Assisted Selection (MAS) was carried out for the waxy
gene located on the short arm of rice chromosome 6 using primers 484
and 485 (Bligh et al. 1995) and for the fertility restorer gene mapped on
the long arm of rice chromosome 10 using tightly linked STMS marker
RM6100 (Prakash 2003). To assess the relative contribution of the two
parental genomes in the segregants and to identify selections with
greater genetic similarity with the recurrent parent, STMS analysis was
carried out. Rice STMS markers were chosen representing all the 12
linkage groups of rice at an interval of 20 cM (Visscher 1996), covering
both the arms of the chromosomes for parental polymorphism survey.
Information regarding chromosomal location and sequence of primers
was obtained from Cho et al. (2000), McCouch et al. (2002), Panaud
et al. (1996) and Temnykh et al. (2000, 2001). The PCR mixture was
prepared and amplification was carried out under standard conditions
(Temnykh et al. 2000). Annealing temperature was adjusted for each
primer pair according to the manufacturers? instruction. The amplified
products were resolved in 3% Metaphor agaroseTM(http://www.
lonza.com) and scored for PB 1 alleles as A and IRBB55 alleles as B
for each primer-genotype combination. The assessment of genomic
contribution of the parents in the elite selections based on STMS
marker data was carried out using the software programme Graphical
Geno Types (GGT) Version 2.0 (Van Berloo 1999).
chromosome segments in the background of the recipient parent in
altered trait expression, association mapping was carried out. First, the
presence of population structure (Q) was analysed using the STRUC-
TURE software (http://pritch.bsd.uchicago.edu/structure.html, Prit-
chard et al. 2000). Of the 12 different alternative structures, one (k2)
that better represented the relationship among the selections and the
parents was chosen. To control the bias due to pedigree relationship,
relative kinship (K) was also estimated by the software SPAGeDi
version 1.2 (http://www.ulb.ac.be/sciences/ecoevol/spagedi.html). The
results of both the above analyses along with the phenotypic data
were used to establish marker-trait association by GLM using
index.html). At R2-Model > 0.6 and P-Marker<0.01, a marker-trait
association was considered significant.
To determine possible role of the donor
Evaluation of agronomic and quality traits:
evaluated for their agronomic performance in a randomized block
design with three replications (five rows per replication) with the
parents as checks during 2003 in the Research Farm of the IARI. The
plants were spaced at 20 · 20 cm in 5 m long rows and were raised
following recommended agronomic practices. Data were recorded for
plant height, tillers/plant, number of effective tillers/plant, panicle
length, number of filled grains/panicle and 1000-grain weight. The
physico-chemical characters such as kernel dimensions of milled and
cooked rice; aroma (Sood and Siddiq 1978) and alkali spreading value
(Little et al. 1958) were analysed as per standard protocols. Amylose
content was estimated adopting colorimetric method (Juliano 1971).
The statistical analysis of the data was performed using MSTAT-C
Further, the evaluation of the agronomic performance of two
selected elite lines was carried out at seven different locations during
2004 in the Basmati growing region of the northwestern India. One of
these selections was promoted to advanced variety trials and thus
further evaluated in 2005 (nine locations) and 2006 (11 locations). At
the Research Farm of IARI, initial screening of the selections for BB
resistance was performed by artificial inoculation using the most
virulent isolate from the Basmati region. Under the National Basmati
Trial, field screening for disease resistance under natural conditions
was carried out at 23 different locations throughout India including
four (Kaul, Haryana; Ludhiana, Punjab; Pantnagar, Uttar Pradesh
and New Delhi) in the Basmati growing region. These locations were
chosen on the basis of occurrence of highly favourable predisposing
factors for the disease. The disease reaction was recorded on a 1–9
scale at maximum tillering stage as per Standard Evaluation System
for Rice (INGER 1996) and the susceptibility index (SI) was computed
as average disease score over the locations. SI score > 6 was
considered as susceptible reaction. Samples for grain and cooking
quality analysis were drawn from the produce of the trial conducted at
rice research station, Kaul, Haryana, a typical Basmati growing
location in India. The grain and cooking quality analysis was carried
out at Directorate of Rice Research, Hyderabad, India and Central
Rice Research Institute, Cuttack, India.
The selected lines were
Foreground selection for BB resistance, waxy and fertility
The presence of both xa13 and Xa21 genes for BB resistance in
the BC1F5generation recombinants was ascertained by using
132Gopalakrishnan, Sharma, Anand Rajkumar, Joseph, Singh, Singh, Bhat, Singh, and Mohapatra
the linked markers (Fig. 1a,b). Restriction digestion of the
1100 bp amplified product from the xa13-linked marker
RG136, gave a 950 bp fragment specific to the susceptible
recipient parent ?PB 1?, and two fragments of size 500 and
450 bp specific to the resistant donor IRBB55 as well as in all
the 31 BC1F5recombinants. An additional 150 bp fragment
was common to both the parents and all the recombinants. For
Xa21, the STS marker pTA248 amplified a resistant parent
specific 950 bp fragment in all the recombinants. These results
thus confirmed the presence of both xa13 and Xa21 in
homozygous condition in all the selected recombinants. In
case of waxy gene, the gene-based microsatellite marker
amplified a 107 bp fragment in ?PB 1? and 120 bp fragment
in IRBB55. All the selections in BC1F5had 107 bp fragment
similar to that of ?PB 1? (Fig. 1c). The molecular analysis with
the fertility restorer gene linked STMS marker RM6100
produced the maintainer specific 150 bp allele in ?PB 1?,
whereas, IRBB55 had a fertility restorer gene specific allele of
175 bp. All the 31 selections in BC1F5possessed the ?PB 1?
specific allele of 150 bp (Fig. 1d).
Background analysis using mapped STMS markers
A set of 270 STMS markers spanning across the 12 chromo-
somes were utilized for surveying polymorphism between
?PB 1? and IRBB55. Of these, sixty-nine markers (25.5%) were
polymorphic. Background analysis of the BC1F5 selections
carried out using these markers showed an average recovery of
75.3% of the PB 1 genome while that of IRBB55 genome was
21.6% with residual heterozygosity of 3.1%. Seven lines had
recovered more than 80% of the PB 1 genome (Table 1).
Chromosome wise analysis of the background showed com-
plete recovery of chromosome 6 from PB 1 in all the
recombinants. For the other chromosomes, at least some
individuals had inherited a segment from the non-Basmati
parent IRBB55. Higher frequency of recombinants (>50%)
carrying donor segments was observed for chromosome 1
(around marker RM237), chromosome 3 (between markers
RM231 and RM2326), chromosome 5 (region surrounding
RM274) and chromosome 12 (region around RM3331). This
was followed by a segment in chromosome 10 (around
RM474) that was inherited in 35.5% of the recombinants.
With regard to rest of the genome, the frequency of the
recombinants with donor segments was low. The chromosome
2 had residual heterozygosity around the locus RM341 in
80.6% of the selections even after five generations of selfing.
Among the selections, Pusa 1460-01-32-6-7-67 was found to
have recovered as high as 86.3% of the PB 1 genome, which
was followed by selection Pusa 1460-01-32-2-2-45 with 82%
?PB 1? genome recovery (Table 1). Pusa 1460-01-32-6-7-67
inherited the chromosomes 1, 3, 6 and 7 completely from the
recurrent parent, short segments from non-Basmati donor
parent for the telomeric ends of chromosomes 4, 9, 10 and 11,
and relatively longer non-Basmati segments for the long arm
of the chromosomes 5, 8 and 12. The short arms of all the
chromosomes except 10 were from the Basmati parent (Fig. 2).
Heterozygosity around the marker RM341 on chromosome 2
was also retained in this line.
Analysis of introgression for the carrier chromosomes
The extent of introgression from the non-Basmati parent for
the chromosomes 8 and 11 that carried the two BB resistance
genes xa13 and Xa21, respectively, was estimated using the
500 bp + 450 bp
33 MM 1
Fig. 1: Marker assisted foreground selection for xa13 (a), Xa21 (b), waxy (c) and fertility restorer gene (d). In (a), HinfI digestion of the 1100 bp
fragment gave 950 and 150 bp fragments in susceptible parent ?PB 1?, but gave 500, 450 and 150 bp fragments in the resistant parent and all the 31
BC1F5selections. The 150 bp fragment, which was common to all is not shown in the figure. In (b), amplification with pTA248 produced a 950 bp
fragment in homozygous resistant parent IRBB55 and in all the 31 selections, while the susceptible parent ?PB 1? had a 650 bp fragment. In (c),
amplification for waxy gene produced a 107 bp fragment in all the selections similar to the recipient parent ?PB 1?, while the donor parent IRBB55
had a 120 bp fragment. In (d), amplification with RM6100 gave a 150 bp fragment in all the selections similar to ?PB 1? whereas in IRBB55 a
175 bp fragment was amplified. Lane M: DNA size standard, Lane 1: ?PB 1?, Lane 2: IRBB55, Lanes 3 - 33: BC1F5selections 1 to 31 in the same
order as in the Table 1
MAS for Bacterial blight resistant recombinants in Basmati rice133
Table 1: Agronomic performance, grain quality, bacterial blight resistance and the percentage of parental genome recovery of the selections
Pusa Basmati 1
±0.11 ± 0.03 ± 0.09
1Awns: P, present; A, absent.
2Grain shape: MS, medium slender; LS, long slender.
3Aroma: VS, very strong; S, strong; M, mild; A, absence.
*Significantly different from recipient parent PB 1 at P ¼ 0.05.
134 Gopalakrishnan, Sharma, Anand Rajkumar, Joseph, Singh, Singh, Bhat, Singh, and Mohapatra
markers linked on either side of the two genes. Physical
locations of the markers were used for analysing the
introgressions in base pairs, which presented four contrasting
situations in case of xa13. First situation was represented by
nine lines, in which a genomic region of <7.8 Mb was
introgressed from IRBB55 (Fig. 3a). In the second situation,
there was a recombination observed in the interval as close as
0.5 Mb (between xa13 and RM447) resulting in an intro-
gression of <1.3 Mb region flanking the xa13 as in two lines
namely Pusa 1460-01-75-6-31-219 and Pusa 1460-01-75-6-13-
130 (Fig. 3b). In the third case, the genome introgrossed in
the chromosome 8 was >7.8 Mb but less than 9.9 Mb in two
of the lines. In rest of the 18 lines, the introgression in the
xa13 genomic region was more than 9.9 Mb. With respect to
Xa21 gene region on chromosome 11, three situations of
introgressions were observed. First, the selection Pusa 1460-
PB 1 genome
Fig. 2: Graphical genotype of the elite selection Pusa 1460-01-32-6-7-67 based on background analysis using the sequence tagged microsatellite
site (STMS) markers
Fig. 3: Analysis of genomic introgression associated with bacterial blight (BB) resistant genes xa13 (a, b) and Xa21 (c, d). For xa13 genomic
region in chromosome 8, (a) presents an introgression less than 7.8 Mb and (b) presents an introgression less than 0.5 Mb. For Xa21 genomic
region in chromosome 11, (c) presents an introgression less than 6.7 Mb and (d) presents an introgression less than 8.1 Mb
MAS for Bacterial blight resistant recombinants in Basmati rice135
01-32-6-7-67 had <6.7 Mb (interval between RM21 and
RM5474) genome linked to Xa21 (Fig. 3c). Second situation
was represented by three selections namely, Pusa 1460-01-32-
2-2-45, Pusa 1460-01-75-6-31-219 and Pusa 1460-01-75-6-13-
130,with 6.7 to 8.1 Mb
remaining 27 lines represented the third situation with a
wider range (8.1–25.4 Mb) of genomic introgressions. Among
all the selections analysed for genomic introgressions for the
donor chromosomes, four selections namely Pusa 1460-01-32-
2-2-45, Pusa 1460-01-32-6-7-67, Pusa 1460-01-75-6-31-219
and Pusa 1460-01-75-6-13-130 were found to have the least
linked region introgression in the genomic regions of xa13
(chromosome 8) and Xa21 (chromosome 11).
introgression (Fig. 3d). The
Phenotypic evaluation and association analysis
The agronomic evaluation trial of 31 BC1F5 families was
conducted at the Research Farm of the Indian Agricultural
Research Institute (IARI), New Delhi and the results are
presented in Table 1. All the entries showed resistance com-
parable to the donor parent IRBB55. The average lesion length
among the selections ranged from 1.5 cm to 2.8 cm (SI 1),
whereas the susceptible parent ?PB 1? showed an average lesion
length of 16.4 cm (SI 9). There was significant difference
between some of the selections and the recurrent parent ?PB 1?
for plant height, panicle length, average number of filled
grains/panicle, 1000-grain weight, yield/plant, hulling %,
milling %, kernel length, kernel length after cooking and
aroma, which suggested contribution of the non-Basmati
parent IRBB55 for these characters. Desirable (as in case of
reduced plant height in nine selections, increased yield/plant in
two, and more number of filled grains/panicle in one of the
selections) and undesirable (reduction in length of the cooked
kernel, and hulling and milling percentage in majority of the
selections) effect of IRBB55 was observed in different selec-
tions. Association analysis carried out after controlling the
bias due to population structure and kinship revealed only
three significant marker-trait linkage at R2-Model > 0.6 and
P < 0.01: RM281 with kernel length after cooking, RM231
with 1000-grain weight and RM237 with grains/panicle
explaining 46.5%, 23.8% and 11.6% of the trait variation,
Performance of selections in multilocation trial
Based on background composition, Basmati grain and cook-
ing quality characteristics and agronomic performance in the
research station trial, two of the selections namely Pusa 1460-
01-32-6-7-67 and Pusa 1460-01-32-2-2-45 were chosen for
multilocation evaluation and tested at seven different locations
in the Basmati growing region of northwestern India during
2004. Pusa 1460-01-32-6-7-67, having the highest recovery of
the ?PB 1? genome and showing superior performance, was
promoted for further multilocation testing in 2005 and 2006.
Table 2 shows the mean performance of this selection in
comparison with the recurrent parent ?PB 1? and the traditional
Basmati cultivar ?Taraori Basmati?, which are used as checks in
Basmati Varietal Trial. The mean yield across locations and
years (during 2004, 2005 and 2006) was at par with the ?PB 1?
and significantly higher than Taraori Basmati. In the agro-
nomic trials conducted at four traditional Basmati growing
locations of India during 2006, it recorded an yield of 4.42 t/ha
with a superiority of 11.9% and 33.53% over the checks ?PB 1?
and ?Taraori Basmati?, respectively. With regard to quality, it
had long slender grains (7.4 mm), desirable kernel length after
cooking (13.7 mm), high ASV (7.0) and preferred intermediate
amylose content (23.6%) based on 3 years data. In the panel
test, it was rated to be strongly scented. More importantly, this
recombinant was found resistant to BB with a susceptibility
index of 3.94 compared to 6.20 in ?PB 1? and 3.97 in the non-
Basmati resistant check variety ?Ajay? based on results from 23
locations, which included both traditional Basmati growing
region and BB hot spots in other parts of India. When the
results from the four traditional Basmati locations were
considered, the SI values for the recombinant, ?PB 1? and
?Ajay? were 3.20, 9.00 and 3.33, respectively. On the basis of
Table 2: Mean performance of the
selection Pusa 1460-01-32-6-7-67 in
the multilocation evaluation trials
in India during 3 years (2004, 2005
Plant height (cm)
Flowering duration (days)
Mean yield (t/ha)
Basmati evaluation trial
(2004, 2005, 2006)
Agronomic trial (2006, at
Head rice recovery (%)
Milled rice kernel length (mm)
Milled rice kernel breadth (mm)
Cooked kernel length (mm)
Kernel elongation ratio
Alkali spreading value
Amylose content (%)
Mean Susceptibility Index
Four Basmati locations only
136Gopalakrishnan, Sharma, Anand Rajkumar, Joseph, Singh, Singh, Bhat, Singh, and Mohapatra
these results, Pusa 1460-01-32-6-7-67 has been released as a
new variety named ?Improved Pusa Basmati 1? for commercial
cultivation in India.
The primary objective of the present study was to integrate
marker assisted background analysis with foreground selection
to identify recombinants in which bacterial blight resistance
from a non-Basmati donor is combined with Basmati quality
traits. The BC1F5lines developed from the cross between a
Basmati variety, ?PB 1? and a non-Basmati line IRBB55 were
utilized. We resorted to a single backcross with the Basmati
parent ?PB 1? that was followed by pedigree selection.
Consequently, majority of the BC1F5lines were found to be
similar to the recurrent parent ?PB 1? with respect to days to
50% flowering, days to maturity and Basmati grain and
cooking quality traits. Unlike the conventional backcross
breeding, the adoption of restricted backcross strategy helped
in retaining useful traits besides BB resistance from the non-
Basmati parent IRBB55. Marker assisted selection enabled
validation for the presence of BB resistance genes namely xa13
and Xa21 in homozygous conditions. Four of these lines, Pusa
1460-01-32-2-2-45, Pusa 1460-01-32-6-7-67, Pusa 1460-01-75-
6-31-219 and Pusa 1460-01-75-6-13-130, inherited erect leaves
and sturdy stem from the non-Basmati parent. The erect flag
leaves are advantageous compared to semi-erect flag leaves in
?PB 1? because they increase the photosynthetic efficiency and
hence enhance the productivity. The sturdy stem derived from
IRBB55 helps in overcoming the problem of lodging of ?PB 1?
under high input conditions. The presence of chalky grains in
?PB 1? is an undesirable character. Pusa 1460-01-32-6-7-67 was
free from chalkiness, a trait inherited from IRBB55. Thirteen
lines also received awnless character from IRBB55, which were
otherwise similar to the recurrent parent ?PB 1? with respect to
other characteristics. The erect flag leaf at the stage of crop
maturity would be useful as an easily scorable marker for
establishing distinctness, uniformity and stability of the elite
The microsatellite marker for the granule bound starch
synthase (waxy) gene was utilized for determining the recurrent
parent genotypic constitution in the selections. Ayres et al.
(1997) demonstrated that the waxy microsatellite is polymor-
phic enough to distinguish most rice cultivars in different
amylose classes, yet stable enough to be easily traced through
multiple generations. They showed that the cultivars having
the (CT)14repeats (ca.120 bp fragment) had 20–23% amylose
content and those in (CT)11class (ca. 107 bp fragment) had
more than 23% amylose content. In our study, the Basmati
and the non-Basmati parents had 24.3% and 21.3% amylose
content (23.45% and 22.54% based on multilocation results),
respectively. All the lines in BC1F5 possessed the (CT)11
microsatellite class and also had >23% amylose content. Thus
MAS for the waxy locus helped recovery of the expected
amylose content in the BC1F5selections.
In any hybrid-breeding program, the availability of heter-
otic combinations of A and R lines is the primary require-
ment. With concerted breeding efforts, an array of Basmati
quality restorers has been developed at the IARI, New
Delhi. However, the cytoplasmic male sterile (CMS) lines
with Basmati quality traits are limited, which is a major
limitation to the development of Basmati rice hybrids. ?PB 1?,
the recurrent parent used in this study, is a perfect
maintainer of WA cytoplasm. In contrast, IRBB55 carries
the fertility restoration ability from its progenitor variety
?IR24?. MAS using the fertility restorer gene linked marker
RM6100 helped in unequivocally establishing the presence of
?PB 1? allele in all the selections. Thus, all these selections
possessing Basmati quality traits along with BB resistance
are potential candidates for conversion into new Basmati
quality CMS lines.
We employed co-dominant STMS markers mapped to
specific chromosomes of rice for background analysis. This
enabled us to determine precise parental genomic contribution
in respect of each chromosome, unlike the random markers
such as AFLP/ISSR, which have been used earlier (Chen et al.
2001, Liu et al. 2003). We identified the elite selection Pusa
1460-01-32-6-7-67 that recovered maximum (86.3%) recurrent
parent genome with donor chromosome segment of <7.8 Mb
in the vicinity of xa13 gene and <6.7 Mb in the region
flanking Xa21 gene. The recovery of recombination event in
the interval as close as 0.5 Mb observed in the xa13 flanking
region in two of the selections also revealed that there exists a
possibility of reducing the linked genomic regions to even
lesser degree. Carrying out selection for the recurrent parent
alleles at the linked flanking marker loci and for the donor
parent allele in respect of the gene under transfer, one can
significantly reduce the length of the linked genomic region
being introgressed from the donor. Linkage drag in backcross
breeding program results in introduction of other linked genes
in addition to the target gene(s), which influences the end
product (Stam and Zeven 1981). In case, the donor genotype is
an unadapted/wild species then the linkage drag might result in
undesirable end product. In our study, although the donor
parent IRBB55 is a near isogenic line developed from ?IR24?,
which is a prominent high yielding rice variety, there was
concern about the efficiency of recovery of the Basmati traits
due to either direct negative effect of this non-Basmati parent
or through indirect negative interactions between the donor
and the recipient genomic regions in the recombinants. In
absence of information on the location of the genes for
Basmati quality traits in the genomic regions linked to Xa21
and xa13, we went for selection against the donor segment in
the linked region. Use of markers linked to the genes under
transfer in background analysis helped reducing the donor
chromosome segments, and thus the linkage drag.
Our efforts to identify additional quantitative trait loci
(QTL) by association analysis had only limited success. The
association of RM281 with kernel length after cooking was
new while the linkage of RM231 with 1000-grain weight (Li
et al. 1995) and RM237 for filled grains per panicle (Tan et al.
1999) was already reported. Additional efforts are therefore,
required to assign possible roles to the genomic regions
introgressed from the non-Basmati donor in the selections. The
results from the background analysis, when correlated with the
earlier published reports, suggested that it enabled recovery of
genomic regions influencing expression of Basmati traits. The
selections having the Basmati quality traits had Basmati parent
alleles for the marker regions reported linked to quality trait
QTL: RM128 region on chromosome 1 for grain length/
breadth ratio (Tsunematsu et al. 1996); RM204 and RM225
region on chromosome 6 for milled rice kernel length and
breadth (Xing et al. 2002); RM44 and RM339 region on
chromosome 8 with aroma and cooked kernel elongation ratio.
Background analysis in conjunction with phenotypic evalua-
tion thus enabled efficient recovery of the recurrent parent
MAS for Bacterial blight resistant recombinants in Basmati rice137
genome along with the Basmati quality traits in some of the
selections. In spite of repeated selfing following a single
backcross, heterozygosity was retained around marker RM341
on the chromosome 2 in a majority of the selections. This
could be due to selective advantage offered in respect of any of
the characteristics selected for including the quality traits. It is
also possible that this region is linked to spikelet fertility in
rice, wherein the homozygote is eliminated as sterile spikelets
in the panicle. Further studies, however, are needed to
understand this genomic region.
In this study, we successfully integrated background analysis
with foreground selection to identify desirable bacterial blight
resistant Basmati type recombinants from a Basmati · non-
Basmati cross. Backcross pedigree strategy facilitated combin-
ing useful traits from both the parents. Our efforts led to
development of new variety ?Improved Pusa Basmati 1?, which
has been released for commercial cultivation in India. This is
the only source of BB resistance now available in the aromatic
germplasm and is being used as a donor for BB in the Basmati
The authors thank the Director, National Research Centre on
Plant Biotechnology for providing all the facilities for molec-
ular work; the Head, Division of Genetics, for the field facility;
and Dr K. V. Prabhu, National Phytotron Facility for his help
in advancing the generations in Phytotron. The first author is
thankful to the Council of Scientific and Industrial Research,
Government of India, for providing financial assistance in the
form of research fellowship.
Ayres, N. M., A. M. McClung, P. D. Larkin, H. F. J. Bligh, C. A.
Jones, and W. D. Park, 1997: Microsatellites and a single nucleotide
polymorphism differentiate apparent amylose classes in an extended
pedigree of US rice germplasm. Theor. Appl. Genet. 94, 773—781.
Beckman, J. S., and M. Soller, 1990: Review: towards a unified
approach to genetic mapping of eukaryotes based on sequence
tagged microsatellite sites. Biotechnology. 8, 930—932.
Bergman, C. J., J. T. Delgado, A. M. McClung, and R. D. Fjellstrom,
2001: An improved method for using a microsatellite in the rice waxy
gene to determine amylose content. Cereal Chem. 78, 257—260.
Bligh, H. F. J., R. I. Till, and C. A. Jones, 1995: A microsatellite
sequence closely linked to the waxy gene of Oryza sativa. Euphytica
Brinkman, M. A., and K. J. Frey, 1977: Yield component analysis of
oat isolines that produce different grain yields. Crop Sci. 17,
Chen, S., X. H. Lin, C. G. Xu, and Q. Zhang, 2000: Improvement of
bacterial blight resistance of ?Minghui 63?, an elite restorer line of
hybrid rice, by molecular marker-assisted selection. Crop Sci. 40,
Chen, S., C. G. Xu, X. H. Lin, and Q. Zhang, 2001: Improving
bacterial blight resistance of ?6078?, an elite restorer line of hybrid
rice, by molecular marker-aided selection. Plant Breed. 120,
Cho, Y. G., T. Ishii, S. Temnykh, X. Chen, L. Lipovich, W. D. Park,
N. Ayres, S. Cartinhour, and S. R. McCouch, 2000: Diversity of
microsatellites derived from genomic libraries and GenBank
sequences in rice (Oryza sativa L.). Theor. Appl. Genet. 100,
Doyle, J. J., and J. L. Doyle, 1990: Isolation of plant DNA from fresh
tissue. Focus 12, 13—14.
Frisch, M., M. Bohn, and A. E. Melchinger, 1999a: Comparison of
selection strategies for marker-assisted backcrossing of a gene. Crop
Sci. 39, 1295—1301.
Frisch, M., M. Bohn, and A. E. Melchinger, 1999b: Minimum sample
size and optimal positioning of flanking markers in marker-assisted
backcrossing for transfer of a target gene. Crop Sci. 39, 967—975.
Erratum in Crop Sci. 39, 1903.
Hospital, F., C. Chevalet, and P. Mulsant, 1992: Using markers in
gene introgression breeding programs. Genetics 132, 1199—1210.
Huang, N., E. R. Angeles, J. Domingo, G. Magpantay, S. Singh,
G. Zhang, N. Kumaravadivel, J. Bennett, and G. S. Khush, 1997:
Pyramiding of bacterial blight resistance genes in rice: marker
assisted selection using RFLP and PCR. Theor. Appl. Genet. 95,
INGER, 1996: Standard Evaluation System for Rice, 4thEd, Inter-
national Rice Research Institute, Los Banos, Manila.
Joseph, M., S. Gopala Krishnan, R. K. Sharma, V. P. Singh,
A. K. Singh, N. K. Singh, and T. Mohapatra, 2004: Combining
bacterial blight resistance and Basmati quality characteristics by
phenotypic and molecular marker-assisted selection in rice. Mol.
Breed. 13, 377—387.
Juliano, B. O., 1971: A simplified assay for milled rice amylose. Cereal
Sci. Today 16, 334—338.
Larkin, P. D., and W. D. Park, 2003: Association of waxy single
nucleotide polymorphisms with starch characteristics in rice (Oryza
sativa L.). Mol. Breed. 12, 335—339.
Li, Z. K., S. R. M. Pinson, J. W. Stansel, and W. D. Park, 1995:
Identification of quantitative trait loci (QTLs) for heading date and
plant height in cultivated rice (Oryza sativa L.). Theor. Appl. Genet.
Little, R. R., G. B. Hilder, and E. H. Dawson, 1958: Differential effect
of dilute alkali on 25 varieties of milled white rice. Cereal Chem. 35,
Liu, S. P., X. Li, C. Y. Wang, X. H. Li, and Y. Q. He, 2003:
Improvement of resistance to rice blast in Zhenshan 97 by molecular
marker-aided selection. Acta Bot. Sin. 45, 1346—1350.
McCouch, S. R., L. Teytelman, Y. Xu, K. B. Lobos, K. Clare,
M. Walton, B. Fu, R. Maghirang, Z. Li, Y. Xing, Q. Zhang,
I. Kono, M. Yano, R. G. Fjellstrom, G. De Clerk, D. Schneider,
S. Cartinhour, D. Ware, and L. Stein, 2002: Development and
mapping of 2240 new SSR markers for rice (Oryza sativa L.). DNA
Res. 9, 199—207.
Openshaw, S. J., S. G. Jarboe, and W. D. Beavis, 1994: Marker
‘‘Analysis of Molecular Marker Data’’, Corvallis, OR. 5–6
Aug.1994, 41–43. Am. Soc. Hortic. Sci., Alexandria,VA, and
SSA, Madison, WI.
Panaud, O., X. Chen, and S. R. McCouch, 1996: Development of
microsatellite markers and characterization of simple sequence
length polymorphism (SSLP) in rice (Oryza sativa L.). Mol. Gen.
Genet. 252, 597—607.
Prakash, P., 2003: Molecular Mapping of Fertility Restorer Gene(s)
and Validation of Rf – Gene Linked Markers in Rice. M.Sc. Thesis.
Indian Agricultural Research Institute, New Delhi, India.
Prathepa, P., and V. Baimai, 2004: Variation of Wx microsatellite
allele, waxy allele distribution and differentiation of chloroplast
DNA in a collection of Thai rice (Oryza sativa L.). Euphytica 140,
Pritchard, J. K., M. Stephens, and P. Donnelly, 2000: Inference of
population structure using multilocus genotype data. Genetics 155,
Ronald, P. C., B. Albano, R. Tabien, L. Abenes, K. Wu,
S. R. McCouch, and S. D. Tanksley, 1992: Genetic and physical
analysis of the rice bacterial blight disease resistance locus, Xa21.
Mol. Gen. Genet. 236, 113—120.
Sanchez, A. C., D. S. Brar, N. Huang, Z. Li, and G. S. Khush, 2000:
Sequence Tagged Site marker-assisted selection for three bacterial
blight resistance genes in rice. Crop Sci. 40, 792—797.
breeding. Proc. Symposium
138Gopalakrishnan, Sharma, Anand Rajkumar, Joseph, Singh, Singh, Bhat, Singh, and Mohapatra
Singh, V. P., E. A. Siddiq, F. U. Zaman, and A. R. Sadananda, 1988:
Improved Basmati donors. Int. Rice Res. Newsl. 13, 22—25.
Singh, S., J. S. Sidhu, N. Huang, Y. Vikal, Z. Li, D. S. Brar,
H. S. Dhaliwal, and G. S. Khush, 2001: Pyramiding three bacterial
blight resistance genes (xa5, xa13 and Xa21) using marker-assisted
selection into indica cultivar PR 106. Theor. Appl. Genet. 102,
Sood, B. C., and E. A. Siddiq, 1978: A rapid technique for
scent determinations in rice. Indian J. Genet. Plant Breed. 38,
Stam, P., and A. C. Zeven, 1981: The theoretical proportion of the
donor genome in near isogenic lines of self fertilizers bred by
backcrossing. Euphytica 30, 227—238.
Takeda, Y., S. Hizukuri, and B. O. Juliano, 1987: Structure of rice
amylopectins with low and high affinities for iodine. Carbohydr.
Res. 168, 79—88.
Tan, Y. F., J. X. Li, S. B. Yu, Y. Z. Xing, and C. G. Xu, 1999: The
three important traits for cooking and eating quality of rice grains
are controlled by a single locus in an elite rice hybrid, Shanyou 63.
Theor. Appl. Genet. 99, 642—648.
Temnykh, S., W. D. Park, N. Ayres, S. Cartinhour, N. Hauck,
L. Lipovich, Y. G. Cho, T. Ishii, and S. R. McCouch, 2000:
Mapping and genome organization of microsatellite sequences in
rice (Oryza sativa L.). Theor. Appl. Genet. 100, 697—712.
Temnykh, S., G. DeClerck, A. Lukashova, L. Lipovich, S. Cartinhour,
and S. R. McCouch, 2001: Computational and experimental
analysis of microsatellites in rice (Oryza sativa L.): frequency, length
variation, transposon associations, and genetic marker potential.
Genome Res. 11, 1441—1452.
Tsunematsu, H., A. Yoshimura, Y. Harushima, Y. Nagamura,
N. Y. Kurata, M. Yano, T. Sasaki, and N. Iwata, 1996: RFLP
framework map using recombinant inbred lines in rice. Breed Sci.
Van Berloo, R., 1999: GGT: Software for display of graphical
genotypes. J. Hered. 90, 328—329.
Visscher, P. M., 1996: Proportion of the variation in genetic compo-
sition in backcross programs explained by genetic markers. J. Hered.
Xing, Z., F. Tan, P. Hua, L. Sun, G. Xu, and Q. Zhang, 2002:
Characterization of the main effects, epistatic effects and their
environmental interactions of QTLs on the genetic basis of yield
traits in rice. Theor. Appl. Genet. 105, 248—257.
Yoshimura, S., R. J. Nelson, A. Yoshimura, T. W. Mew, and
N. Iwata, 1992a: RFLP mapping of the bacterial blight resistance
genes Xa-3 and Xa-4. Rice Genet. Newsl. 9, 136—138.
Yoshimura, S., A. Yoshimura, A. Saito, N. Kishimoto, M. Kawase,
M. Yano, M. Nakagraha, T. Ogawa, and N. Iwata, 1992b: RFLP
analysis of introgressed segments in three near-isogenic lines of rice
for bacterial blight resistance genes, Xa-1, Xa-3 and Xa-4. Jpn.
J. Genet. 67, 29—37.
Yoshimura, S., A. Yoshimura, N. Iwata, S. R. McCouch, M. L.
Abenes, M. R. Baroidan, T. W. Mew, and R. J. Nelson, 1995a:
Tagging and combining bacterial blight resistance genes in rice using
RAPD and RFLP markers. Mol. Breed. 1, 375—387.
Yoshimura, S., A. Yoshimura, R. J. Nelson, T. W. Mew, and
N. Iwata, 1995b: Tagging Xa-1, the bacterial blight resistance gene
in rice, by using RAPD markers. Jpn. J. Breed. 45, 81—85.
MAS for Bacterial blight resistant recombinants in Basmati rice139