Euphytica (2024) 220:60

https://doi.org/10.1007/s10681-024-03331-4

RESEARCH

Marker‑assisted gene introgression for resistance

to Xanthomonas oryzae pv. oryzae in rice for the control
of bacterial leaf blight
Md. Ariful Islam · Md. Moniruzzaman Hasan · Md. Ataur Rahman ·
Tanbin Akter · Md. Ashraful Haque

Received: 22 February 2023 / Accepted: 5 March 2024

© The Author(s), under exclusive licence to Springer Nature B.V. 2024

Abstract Bacterial leaf blight, sometimes known as
BLB, is one of the most damaging diseases that may
jeopardize the world’s supply of rice. It is caused by
the bacterium Xanthomonas oryzae pv. oryzae (Xoo).
It has caused sharp decline in production in regions of
the globe that produce rice. More than 40 previously
characterized resistance (R) genes enable host toler-
ance for diverse Xoo strains. In this study, three BLB
resistant genes, including xa5, xa13, and Xa21, which
have been crucial in disease prevention in Bangla-
desh, were introgressed into populations. These popu-
lations were created by crossing IRRI 154, a popu-
lar rice variety with a modern genetic background,
with IRBB66 (resistant to BLB). Fifteen virulent
bacterial isolates were used for BLB infection, and
promising recombinants from the F ­ 5 and B
­ C2F4 gen-
erations were found to be resistant. Using marker
assisted selection (MAS) with gene-specific primers
on generations ­F5 and ­BC2F4, we were able to find
that 60 recombinant introgressed lines (RILs) had
M. A. Islam · M. M. Hasan · M. A. Rahman · T. Akter
Advanced Seed Research and Biotech Centre, ACI
Limited, Dhaka 1212, Bangladesh
M. M. Hasan (*)
Pest Management Division, Bangladesh Jute Research
Institute, Dhaka 1207, Bangladesh
e-mail: hasan_vi@yahoo.com
M. A. Haque
Department of Genetics and Plant Breeding, Bangladesh
Agricultural University, Mymensingh 2202, Bangladesh
a pyramiding of BLB resistance genes. In terms of
agronomic performance, the RILs outperformed both
their donor and recipient parents, demonstrating that
the RILs had pyramided three resistance genes, there-
fore conferring broad-spectrum BLB resistance. The
produced BLB-resistant RILs offer substantial future
development potential, either as cultivable crops or as
BLB resistance donor material for use in boosting the
yields of other rice lines.
Keywords Bacterial leaf blight · Gene
introgression · Gene pyramiding · Oryza sativa L
Introduction
Rice (Oryza sativa L.), which is the most widely
grown cereal grain, is important for getting rid of
hunger. It is cultivated on around 160 million hec-
tares and produces approximately 502.7 million tons
(Prasad et al. 2017). In Bangladesh, rice is the most
widely farmed crop, and more than 90% of the popu-
lation depends on it for sustenance. Most people get
76% of their daily calories and 66% of their protein
from rice (Bhuiyan et al. 2002). The rice industry
accounts for 1/6 of national revenues and 50% of agri-
cultural GDP (BBS 2016). Bangladesh cultivated rice
over a total area of 11.83 million hectares, harvesting
a total of 35.85 million metric tons with an average
yield of 4.55 metric tons per hectare (USDA 2020).

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Industrialization and urban sprawl are reduc-
ing arable land while the population continues to
increase. Due to the rise in global population and
economic growth, farmers must produce more rice
on fewer acres. Currently, farmers can harvest around
5 tons per hectare, although the production potential
of rice is approximately 10 tons per hectare (Khush
and Jena 2009). Rice consumption is expanding at an
alarming pace; thus, rice output must rise by more
than 40% by 2030 to fulfil the rising demand for
rice (Khush 2005). Thus, rice yield enhancement is
the primary foundation of food security (Suela et al.
2019).
In addition, rice production is regularly hampered
by biotic and abiotic stresses. One of the most damag-
ing bacterial diseases of rice in the world is Bacterial
Leaf Blight (BLB), which is brought on by the vas-
cular pathogen Xanthomonas oryzae pv. oryzae (Xoo)
(Luo et al. 2016; Sundaram et al. 2011). Depending
on the variety, the severity and stage of infection,
and the environment, the disease might cut yields by
as much as 50% in severe cases (Alam et al. 2016;
Shankara et al. 2017). In recent years, new races of
this pathogen have been identified in Bangladesh,
causing a significant yield reduction (50–70%) among
irrigated types (Islam et al. 2016).
A vascular pathogen called Xanthomonas infects
the rice plant systemically and causes lesions in the
veins of the leaf blades that may extend to the sheath
and range in colour from yellow to tan to grey to
white (Sundaram et al. 2011). According to Laha
et al. (2009), infected crop grains had poor water
absorption, volume expansion, and kernel elongation,
which decreased their market value. About 20–40%
of the yield was reduced because of a massive infec-
tion during the peak of tillering (Mew 1987).
Both rainfed and irrigated rice farming regions
across the globe experience varying degrees of bacte-
rial leaf blight disease each year (Shankara et al. 2017).
The occurrence and progression of diseases are signifi-
cantly influenced by topographic and climatic factors.
Discrete light and dryness have an impact on the causal
organism. The disease is more common in regions that
get more than 200 mm of rainfall per month, and lesion
formation occurs between 25 and 30 °C. The overuse of
fertilizer is an important agricultural practice that helps
bacterial blight spread (Cha et al. 1982). The severity
of the infection is also influenced by the density of rice
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Euphytica (2024) 220:60
growing; it is more severe in wider-spaced situations
with consistent nitrogen levels (Jabeen et al. 2012).
The devastating effects of BLB on rice make the
research and development of pest management strate-
gies imperative. BLB is managed through a variety of
techniques that limit initial inoculum and subsequent
pathogen growth on host plants. Cultural practices,
biological control, and chemical control are ineffec-
tive when the disease reaches epidemic levels (Bha-
rani et al. 2010; Laha et al. 2009), and no commer-
cially available bactericide is effective against BLB
(Lee et al. 2003). Reviewing the severity and exten-
sive harm caused by this devastating disease on a
global scale, the experts focused their attention on its
management and control via the adoption of resistant
varieties. Host plant resistance has shown the greatest
increase in suppressing BLB (Gautam et al. 2015).
The fundamental objective of today is to gener-
ate better varieties that are resilient to biotic and abi-
otic stresses (Hasan et al. 2015). Therefore, in order
to increase rice yield, rice breeders must consider
time and money-saving breeding techniques. Tradi-
tional breeding techniques take ten to twelve years
to produce improved varieties (Collard and Mackill
2008). But marker-assisted breeding (MAB) offers
an answer to any problem that arises in rice breeding.
The issues with traditional breeding are resolved, and
breeding costs are decreased by using marker-assisted
breeding in rice. It is a successful, affordable, and
environmentally responsible method for pyramiding
all stress-resistant genes in a genotype, resulting in
host resistance (Das and Rao 2015). Therefore, the
most effective and cost-efficient way of managing
BLB is via the utilization of the natural disease resist-
ance provided by MAB. In this study, phenotypic
and molecular markers were employed to find BLB-
resistant RILs from a cross between the rice varieties
‘IRRI 154’ and ‘IRBB66’ which had the xa5, xa13,
and Xa21 genes.
Materials and methods
Plant materials
In the breeding program for bacterial leaf blight
resistant line development, the crossing pro-
gram started in 2017. A total of 31 parent lines
were assessed for successful crossing based on the
Euphytica (2024) 220:60
screening findings, which included gene-specific and
linked markers in order to identify the key bacterial
blight resistance gene, xa13. Finally, a cross between
IRRI 154 and IRBB66 was selected for advancement.
Individuals of the F ­ 1 population were used to detect
the xa13 gene and named ASRBCR 1077 (T. Aman
2018). Then, ­F1 plants were successfully crossed with
recurrent parent IRRI 154 to produce ­BC1F1 progeny
to increase the recipient parent’s characteristics. The
following year, in T. Aman 2019, B ­ C1F1 plants were
again crossed with the recurrent parent IRRI 154 to
create ­BC2F1 progeny, which were named ASRBCR
1077:15:7, in order to enhance the recipient parent’s
characteristics. For generation advancement, ­BC2F1
plants were salved to create ­BC2F2 progeny.
In screen house Rapid Generation Advance
(RGA), we plant ­ BC2F2 population (ASRBCR
1077:15:7-B) for generation advancement without
following any season in 2019. After completing two
RGA cycles in T. Aman 2020, we got ­BC2F4 genera-
tions of ASRBCR 1077:15:7-B-B RGA-B RGA and
these are preserved for Line Stage Trial (LST) at the
next Boro 20–21 season.
In field Rapid Generation Advance (RGA), segre-
gating populations of ASRBCR 1077-B were planted
with extremely tight spacing (5 cm × 5 cm) and very
little fertilizer in 9 m by 1.25 m raised beds (Fig. 1).
A high density of 3000–4000 randomly chosen
­F2 seeds were sown in raised beds. The growth of
healthy seedlings was made possible by using a lower
seed rate (50 g ­m−2) than typical. With split applica-
tions of N at 15, 30, and 50 days after transplanting
(DAT), fertilizers at 60:9.5:30:10:1.8 kg NPKSZn/ha
(130-48.5-60-55-5.5 kg/ha Urea-TSP-MoP-Gypsum-
ZnSO4) were employed. At the time of the last stage
of land preparation, a total quantity of P K S was
applied. To protect against rodent damage, a poly-
thene fence was put up around the plants. To mitigate
biotic stress, necessary control measures were also
employed. When the panicle held more than 50% ripe
seed, it was collected. After completing third RGA
cycle in T. Aman 2020, we got ­F5 generations of
ASRBCR 1077-B-B RGA-B RGA-B RGA and these
are preserved for Line Stage Trial (LST) at the next
Boro 20–21 season.
Twelve advanced backcross ­ (BC2F4) generations
and forty-eight advanced forward ­(F5) lines of IRRI
154/IRBB66 were developed by using IRRI 154 as
the recurrent parent, the recessive xa13 and dominant
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Fig. 1  Pictorial view of Rapid generation advancement
Xa21 genes from donor IRBB66 were transmitted
to the backcross (BC) population and forward lines
with the recessive xa5 gene. These advanced lines
were examined at the Advanced Seed Research and
Biotech Centre in Dhaka, Bangladesh, to see whether
they demonstrated resistance to bacterial leaf blight.
As positive control, IRBB66 from the International
Rice Research Institute (IRRI) was used because it
was known to have genes that protect it from bacterial
leaf blight.
Preparation of bacterial inocula
Fifteen virulent isolates of bacterial blight were col-
lected from different AEZs of Bangladesh. These
isolates were cultured in peptone sucrose agar (PSA)
medium slant for 72 h at 30 °C. The bacterial suspen-
sion concentration was adjusted to 3.3 × ­108 CFU/ml
by adding a total of 10 ml of sterile distilled water to
the slant and measuring the optical density (OD) at
600 nm using a spectrophotometer (WA-60DT plus,
Wave Analytics Germany). An OD600 of 1 corre-
sponds to a bacterial cell number of 3.3 × ­108 CFU/
ml, the optimal concentration for causing BLB dis-
ease in rice (Rashid et al. 2021).
Rice plant inoculation using bacterial isolates
All RILs, their parents, and the check varieties had
their seeds soaked in a 500 ppm solution of streptocy-
cline for eight hours to inhibit seed transfer. Earthen
pots (20 cm in diameter) were sown with clean seeds
before being put in the greenhouse. The modified
clipping procedure (Kauffman 1973) was used to
inoculate 45-day-old seedlings with a bacterial isolate
suspension in a double-door net house. In brief, each
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bacterial blight isolate inoculated twelve leaves per DNA isolation

entry, which were then trimmed from the tip at the
maximum of tillering. The scissors were disinfected
by being rinsed three times in sterile water and then
stored in 70% ethanol for 30 min before being dipped
into the fresh isolate solution. The development of
symptoms was monitored in the inoculated plants. To
demonstrate Koch’s postulates, the bacteria were once
again isolated from the artificially infected seedlings
and contrasted with the original culture.
All the BC populations were grown in the field. After
15 days of transplant, collect the leaf samples and
isolate the DNAs. The DNA extraction procedure fol-
lowed the method proposed by Zheng et al. (1995).
UNICO SQ-4802 double beam scanning spectro-
photometer was used to quantify the concentration
of isolated DNA from the ratio of UV absorbance
at 260 nm and 280 nm. A working solution was pre-
pared maintaining the concentration at 50 ng/µl.

Disease scoring DNA markers and target genes

Following the guidelines suggested by Ke et al.
(2017), disease scoring was done two weeks after
inoculation. Lesion length was measured to provide a
disease score (Table 1). The lesion length is the dis-
tance between the inoculation point and the margin of
the fully blighted middle veins on the leaf.
Table 1  Scale for measuring resistance to bacterial leaf blight
Scale Lesion Length (cm) Description
The xa5 and xa13 recessive genes and the Xa21 dom-
inant gene were selected for the MAS study because
they provide resistance to different BLB races. Using
3 INDEL markers, these genes were detected in order
to enhance BLB resistant materials for rice breed-
ing in BC populations. These indel markers for BLB
resistance were provided by the International Rice
Research Institute (IRRI) as part of the ACI-IRRI
PPP (Table 2). Initially, primer M440 was used to
detect the xa5 gene by looking for a banding pattern
at 158 bp. Similarly, primers M478Lm for xa13 and
M486Lm for Xa21 were used for detecting the pres-
ence of genes.

0 (Immune) < 0.5 cm
1 0.5–3.0 cm
3 3.1–5.0 cm
5 5.1–10.0 cm
7 10.1–15.0 cm
9 > 15.0 cm
Highly resistant (HR) PCR amplification and visualization
Resistant (R)
Moderately resistant (MR) PCR was performed using 10 ng of genomic DNA
Moderately susceptible per 20 µl in a reaction mixture containing 10 mM
(MS)
Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM M ­ gCl2, 0.8
Susceptible (S)
units of Taq polymerase, 200 µM each of dNTPs, and
Highly susceptible (HS)
0.20 µM of primer. Following a 2 min denaturation

Table 2  INDEL markers used for PCR analysis

INDEL marker Sequence Target QTL Chromosome
Number

M440 F 5’- GAC​TGA​CTT​GAC​GAC​TTG​ACG​GGC​TGA​ACT​CTT​TGA​TTA​ xa5 5

TCC​TA -3’
R 5’- GAC​TGA​CTT​GAC​GAC​TTG​ACC​AAC​CCA​TAC​AGA​ATT​TCG​
AGG​AGTG -3’
M478Lm F 5’- TCA​GAG​TGG​AAA​AGA​AAT​ATC​AAG​CAC​AAG​A -3’ and xa13 8
5’- TTT​GGA​CTT​GAG​ATT​TGG​TGA​GAA​TGTAT -3’
R 5’- GAC​CTT​GGC​CAT​GGC​TCA​GTGTT -3’
M486Lm F 5’- ATC​GAA​TTA​TGG​GTG​TTT​TCT​GCT​CTA -3’ Xa21 11
R 5’- CAT​GGT​CAA​TGT​AAG​CTC​GTG -3’ and
5’- TTT​CAT​GGT​CAA​TGC​AAG​TTCT -3’

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session at 94 °C, 30 cycles of denaturing for 1 min at
94 °C, annealing for 1 min at 55 °C, and extending for
2 min at 72 °C were conducted. The last 5 min should
be spent at 72 °C for final extension. After electropho-
resis on 10% polyacrylamide gel for INDEL marker
products, polymorphisms in PCR results were found.
For the xa5, xa13, and Xa21 genes, electrophoresis on
10% polyacrylamide gel was used to score the exist-
ence of a resistant or susceptible band for a specific
gene.
Results
Gene introgression
Using linked and gene-specific markers, 24 resist-
ant progenies were selected against the key bacterial
blight resistance gene, xa13. Then, F ­ 1 plants were
crossed with recurrent parent IRRI 154 to produce
­BC2F1 progeny to enhance the recipient parent’s
characteristics.
Line development using rapid generation advance
technique
To shorten the breeding cycle, using cutting edge
breeding technique rapid generation advance to make
it feasible to get from ­BC2F2 to ­BC2F4 generations in
just one year. After ­BC2F4 generations, at Line Stage
Trial, a total of 392 RILs consisting of ASRBCR
1077-B-B RGA-B RGA-B RGA (192 lines) and
ASRBCR 1077:15:7-B-B RGA-B RGA (200 lines)
were selected on the basis of desired traits like homo-
geneity, plant height, growth duration, grain type, dis-
ease tolerance, and phenotypic acceptability.
Agronomic performance of pyramided lines
In T. Aman 2021, nine lines were chosen from sixty
lines in observational yield trial (OYT) based on
their PAcp (Phenotypic Acceptance) maturity score
(Table 3). In all of these lines, QTL xa5, xa13, and
Xa21 validated the molecular findings on the BLB-
tolerant gene. Compared to plant height, grain pro-
duction, and duration 10 days earlier than IRRI 154
(Table 3), nine lines out of sixty were chosen for the
Advance Yield Trial and artificial screening. Here,
we can see that ASRBCR 1077-B RGA-B RGA-B
Page 5 of 15 60
RGA-182-3 (Table 3) had the best yield compared to
the check variety IRRI 154 with the same length of
time to grow.
Performance of advance lines on the yield and yield
contributing attributes
Variety exhibited significant influence on the yield
contributing characters and yield of the RILs
(Table 4). Plant heights at maturity of the RILs
showed significant variation. The highest plant height
(151 cm) was observed in ASRBCR 1077-B-B
RGA-B RGA-B RGA-182-3 and the lowest (110 cm)
in ASRBCR 1077-B-B RGA-B RGA-B RGA-63-3
(Fig. 2). Results showed that the growth duration
ranged from 102 to 130 days. Maximum yield (6.79
t/ha) was obtained from ASRBCR 1077-B-B RGA-B
RGA-B RGA-7-2. This variation might be due to
heredity, which is directly related to the genetic char-
acteristics of varieties.
Response of RILs to BLB races
In T. Aman 2021, we want to examine the response
of nine RILs (which were selected from 60 RILs for
significantly higher yields than checks) to bacterial
blight. Among nine advance lines with two parent
lines (IRBB66 and IRRI 154), five lines were found
to be resistant against 15 Xoo bacterial isolates after
screening with artificial inoculation of bacterial leaf
blight.
Based on lesion length, out of the 11 lines (includ-
ing 2 parental lines), three lines (ASRBCR 1077-
B-B RGA-B RGA-B RGA-7-2, ASRBCR 1077-B-B
RGA-B RGA-B RGA-141-2 and ASRBCR 1077-B-B
RGA-B RGA-B RGA-184-2) were found to be highly
resistant, Two of them ASRBCR 1077-B-B RGA-B
RGA-B RGA-63-3 and ASRBCR 1077-B-B RGA-B
RGA-B RGA-105-1 were moderately resistant and
rest of four (ASRBCR 1077-B-B RGA-B RGA-B
RGA-15-1, ASRBCR 1077-B-B RGA-B RGA-B
RGA-182-3, ASRBCR 1077-B-B RGA-B RGA-B
RGA-24-1 and ASRBCR 1077:15:7-B-B RGA-B
RGA-70-1) showed moderately sensitive when com-
pared to resistant check IRBB66 (0.68 cm) lesion
length (Table 5).
ASRBCR 1077:15:7-B RGA-B RGA-7-2 is
regarded as the highest resistant since this had the
shortest average lesion length (1.25 cm) with yellow
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Table 3  Gross morphology of advance lines in Observational Yield Trial (OYT) T. Aman 2021
Entry Designation PAcp PH Duration ET PL Yield 1000gwt (gm)

1 ASRBCR 1077-B RGA-B RGA-B RGA-7-2 1 123 109 9 31.0 6.19 26.9427
2 ASRBCR 1077-B RGA-B RGA-B RGA-13-2 7 123 133 10 30.0 4.75 30.2767
3 ASRBCR 1077-B RGA-B RGA-B RGA-14-1 7 134 130 10 29.0 4.52 27.9845
4 ASRBCR 1077-B RGA-B RGA-B RGA-15-2 3 139 106 9 29.7 5.73 25.4827
5 ASRBCR 1077-B RGA-B RGA-B RGA-16-1 9 164 131 10 31.0 5.07 23.1027
6 ASRBCR 1077-B RGA-B RGA-B RGA-17-1 7 123 137 11 29.0 5.05 29.2832
7 ASRBCR 1077-B RGA-B RGA-B RGA-18-1 7 125 130 9 24.0 4.52 27.3476
8 ASRBCR 1077-B RGA-B RGA-B RGA-21-2 7 126 136 10 28.3 5.06 25.3382
9 ASRBCR 1077-B RGA-B RGA-B RGA-22-3 7 128 129 10 28.5 4.11 26.2081
10 ASRBCR 1077-B RGA-B RGA-B RGA-24-1 5 139 103 11 28.7 5.78 22.4197
11 ASRBCR 1077-B RGA-B RGA-B RGA-27-2 7 125 133 12 24.3 5.07 24.0026
12 ASRBCR 1077-B RGA-B RGA-B RGA-29-3 7 118 125 11 28.2 3.78 26.614
13 IRBB66 5 126 129 12 29.3 5.58 25.8766
14 IRRI 154 5 113 123 12 30.5 5.63 25.1142
15 ASRBCR 1077-B RGA-B RGA-B RGA-35-3 7 112 122 13 29.0 3.96 26.931
16 ASRBCR 1077-B RGA-B RGA-B RGA-51-2 7 90 95 9 28.0 2.46 22.3227
17 ASRBCR 1077-B RGA-B RGA-B RGA-53-1 9 140 121 11 31.2 3.52 22.4534
18 ASRBCR 1077-B RGA-B RGA-B RGA-56-1 7 111 133 9 29.7 3.85 27.3571
19 ASRBCR 1077-B RGA-B RGA-B RGA-58-3 7 122 131 8 29.0 4.49 29.3002
20 ASRBCR 1077-B RGA-B RGA-B RGA-63-3 5 110 102 12 30.3 5.86 25.4742
21 ASRBCR 1077-B RGA-B RGA-B RGA-64-2 7 113 135 10 29.7 4.53 26.0869
22 ASRBCR 1077-B RGA-B RGA-B RGA-66-3 7 113 105 7 29.3 3.60 24.5466
23 ASRBCR 1077-B RGA-B RGA-B RGA-70-1 7 145 124 10 30.0 4.82 26.0696
24 ASRBCR 1077-B RGA-B RGA-B RGA-78-2 7 113 132 10 29.0 4.91 29.3458
25 ASRBCR 1077-B RGA-B RGA-B RGA-84-3 7 116 132 12 28.7 4.84 28.2872
26 ASRBCR 1077-B RGA-B RGA-B RGA-103-1 7 131 140 8 31.0 4.95 25.4362
27 ASRBCR 1077-B RGA-B RGA-B RGA-104-1 7 115 135 9 29.0 3.82 25.5293
28 ASRBCR 1077-B RGA-B RGA-B RGA-105-1 5 126 111 7 29.0 6.21 26.0849
29 ASRBCR 1077-B RGA-B RGA-B RGA-107-3 7 136 132 8 29.0 4.76 26.873
30 ASRBCR 1077-B RGA-B RGA-B RGA-108-2 7 133 144 7 29.7 4.42 25.9238
31 ASRBCR 1077-B RGA-B RGA-B RGA-110-2 7 123 135 9 28.0 4.66 21.434
32 ASRBCR 1077-B RGA-B RGA-B RGA-115-3 7 121 129 9 27.7 3.55 27.4779
33 ASRBCR 1077-B RGA-B RGA-B RGA-118-3 7 119 100 10 31.0 3.97 22.0245
34 ASRBCR 1077-B RGA-B RGA-B RGA-119-1 7 112 122 10 30.3 3.92 25.2182
35 ASRBCR 1077-B RGA-B RGA-B RGA-138-1 7 106 132 9 28.7 3.61 26.9278
36 ASRBCR 1077-B RGA-B RGA-B RGA-139-2 7 110 136 11 28.7 4.63 29.0037
37 ASRBCR 1077-B RGA-B RGA-B RGA-140-2 7 113 135 9 29.7 4.16 28.6022
38 ASRBCR 1077-B RGA-B RGA-B RGA-141-2 3 121 111 11 29.7 5.72 25.2446
39 ASRBCR 1077-B RGA-B RGA-B RGA-142-1 7 126 139 9 24.7 4.92 27.9512
40 ASRBCR 1077-B RGA-B RGA-B RGA-145-3 9 142 130 11 26.9 3.84 23.8506
41 ASRBCR 1077-B RGA-B RGA-B RGA-147-2 7 104 95 8 26.0 3.12 27.6699
42 ASRBCR 1077-B RGA-B RGA-B RGA-148-3 7 130 105 9 29.7 3.16 28.929
43 ASRBCR 1077-B RGA-B RGA-B RGA-149-3 7 134 108 9 31.3 3.34 26.9091
44 ASRBCR 1077-B RGA-B RGA-B RGA-170-2 7 106 111 10 28.3 3.22 23.5847
45 ASRBCR 1077-B RGA-B RGA-B RGA-175-3 9 138 100 12 28.0 3.05 21.996
46 ASRBCR 1077-B RGA-B RGA-B RGA-176-1 7 118 128 9 29.7 3.66 28.0254

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Table 3  (continued)
Entry Designation PAcp PH Duration ET PL Yield 1000gwt (gm)

47 ASRBCR 1077-B RGA-B RGA-B RGA-182-3 3 149 104 10 29.7 6.31 23.506
48 ASRBCR 1077-B RGA-B RGA-B RGA-183-1 7 106 95 10 28.0 2.73 23.4828
49 ASRBCR 1077-B RGA-B RGA-B RGA-184-2 5 116 104 11 27.0 6.13 27.0446
50 ASRBCR 1077-B RGA-B RGA-B RGA-191-1 7 110 122 11 28.3 3.79 24.8131
51 ASRBCR 1077:15:7-B RGA-B RGA-1-3 7 118 123 11 28.3 4.21 21.2382
52 ASRBCR 1077:15:7-B RGA-B RGA-3-1 7 107 124 11 24.3 4.04 21.5968
53 ASRBCR 1077:15:7-B RGA-B RGA-24-2 7 122 139 8 28.0 4.74 26.796
54 ASRBCR 1077:15:7-B RGA-B RGA-38-2 7 123 127 11 28.3 4.64 22.7898
55 ASRBCR 1077:15:7-B RGA-B RGA-49-2 7 125 129 10 28.7 4.89 26.8702
56 ASRBCR 1077:15:7-B RGA-B RGA-50-1 7 108 115 7 29.7 3.11 27.7005
57 ASRBCR 1077:15:7-B RGA-B RGA-70-1 3 144 104 10 30.3 5.99 26.3905
58 ASRBCR 1077:15:7-B RGA-B RGA-72-1 7 111 108 11 28.0 3.14 23.5996
59 ASRBCR 1077:15:7-B RGA-B RGA-74-1 7 127 129 11 28.3 4.20 25.7633
60 ASRBCR 1077:15:7-B RGA-B RGA-107-1 9 140 123 11 29.7 4.90 20.7614
61 ASRBCR 1077:15:7-B RGA-B RGA-151-1 7 116 124 11 29.7 4.66 24.4181
62 ASRBCR 1077:15:7-B RGA-B RGA-157-1 7 124 115 10 29.7 4.06 26.3766
Legend: PAcp Phenotypic acceptance, PH Plant height, ET Effective tiller, PL Panicle length, gwt Grain weight

Table 4  Gross morphology of advance lines in Advanced Yield Trial (AYT) during T. Aman 2022
Sl No Designation Height Duration Yield Disease reaction

1 ASRBCR 1077-B RGA-B RGA-B RGA-182-3 151 a 106 b 6.42 a Moderately sensitive
2 ASRBCR 1077:15:7-B RGA-B RGA-70-1 147 a 104 b 6.53 a Moderately sensitive
3 ASRBCR 1077-B RGA-B RGA-B RGA-7-2 123 c 110 b 6.79 a Highly Resistant
4 ASRBCR 1077-B RGA-B RGA-B RGA -15-2 140 ab 106 b 4.33 b Moderately sensitive
5 ASRBCR 1077-B RGA-B RGA-B RGA -141-2 124 c 110 b 5.29 ab Highly Resistant
6 ASRBCR 1077-B RGA-B RGA-B RGA -24-1 141 a 105 b 5.90 a Moderately sensitive
7 ASRBCR 1077-B RGA-B RGA-B RGA -63-3 110 c 102 b 6.12 a Resistant
8 ASRBCR 1077-B RGA-B RGA-B RGA -105-1 124 c 111 b 6.13 a Resistant
9 ASRBCR 1077-B RGA-B RGA-B RGA -184-2 118 c 105 b 6.23 a Highly Resistant
10 IRRI 154(ck) 112 c 125 a 5.98 a Sensitive
11 IRBB66(ck) 124 bc 130 a 5.27 ab Resistant
LSD(0.05) 16.1 11.7 1.53
%CV 7.52 6.38 15.6

to brown color lesion (Fig. 3). The lesion length
was the longest on the advance line, ASRBCR
1077-B-B RGA-B RGA-B RGA-70-1 (11.82 cm)
with grayish color. There was a noticeable differ-
ence in the severity of the symptoms on one parent
recipient line, IRRI 154, where a large leaf lesion
measuring 17.58 cm was found with grayish color
(Table 6).
Molecular marker analysis for characterizing the
RILs
Overall, 392 RILs including 2 parent lines, were phe-
notypically identified for confirmation of 3 distinct
genes (xa5, xa13, and Xa21). Different combina-
tions of multiple-resistance (single, double and triple)
genes were found in these advanced lines. For this,

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Fig. 2  Yield performance of different RILs
the BLB resistant donor parent “IRBB66” provided a
PCR amplicon similar to the resistance line, whereas
the receiver parent “IRRI 154” produced a suscepti-
bility amplicon. In the beginning, primer M440 was
utilized to find the banding pattern at 158 bp to detect
the xa5 gene and its sample size was 394 where
obtained all positive samples. Secondarily, the xa13
gene primer M478Lm identifies the resistance band-
ing pattern at 450 bp as IRBB66 in 73 RILs (61 RILs
from ASRBCR 1077 and 12 RILs from ASRBCR
1077:15:7). And finally, the resistance check IRBB66
verified the 980 bp banding pattern identified by a
PCR marker linked to the Xa21 gene (Chunwongse
et al. 1993).
Discussion
More than half of the people in the world eat rice
every day to meet their nutritional needs. Several
studies have shown that by 2050, the world’s rice sup-
ply would need to increase by a fourfold amount to
keep up with the growing demand for rice (Ray et al.
2013). The quantity and quality of rice crop yields
are also impacted by a variety of biotic and abiotic
stresses. The development of high-yielding cultivars
with added genes for disease resistance is essential for
overcoming these challenges and increasing global
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rice output. Broad-spectrum resistance to BLB,
which is caused by the Xoo bacterium, is particularly
difficult to develop due to the presence of several
genetically varied virulent Xoo strains in various rice-
growing regions across the globe. This makes it par-
ticularly difficult to develop BLB-resistant rice (Das
et al. 2018). The creation of multiple-race-resistant
cultivars has not received much written attention.
Three BLB resistance genes (xa5, xa13, and Xa21)
were put into an IRRI 154 × IRBB66 progeny to make
rice resistant to more than one BLB race. This was
done to make rice farming more sustainable. Three
dominant markers showed PCR amplification in rela-
tion to the BLB resistance genes in IRBB66 (donor)
parent material (Figs. 4, 5, and 6).
The major purpose of this work was to identify
and show the interaction between BLB races and
RILs ­(F5 and ­BC2F4) having three BLB resistance
genes (xa5, xa13, and Xa21). The crossing pro-
gram started in 2017. After the successful crossing,
24 resistant progenies were chosen based on the
screening results in order to identify the essential
bacterial blight resistance gene, xa13. A band at
500 bp was seen in all 24 F ­ 1 progenies, indicating
the existence of a resistance gene. Individuals of the
­F1 population were used to detect the xa13 gene and
named ASRBCR 1077 (T. Aman 2018). Then, ­F1
plants were crossed with recurrent parent IRRI 154
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(2024) 220:60

Table 5  Disease reaction of 9 advance lines with 2 check varieties

Designation ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR IRBB66 IRRI 154
Isolates 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077:15:7-B
RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B
RGA-B RGA- RGA-B RGA- RGA-B RGA- RGA-B RGA- RGA-B RGA- RGA-B RGA- RGA-B RGA- RGA-B RGA- RGA-70-1
105-1 141-2 15-1 182-3 184-2 24-1 63-3 7-2

Xoo1 R R R MS R MR R R MR R MS
Xoo5 R R R R MR MR R HR R MR S
Xoo7 R MR MS R R S R R MS MR S
Xoo9 R R R R R R R R R R S
Xoo15 R HR R MR R R R R R R MR
Xoo16 R R MR R R R MR R R R S
Xoo17 MS R S MS R MS MR MR MS R HS
Xoo18 R R R R R R R R R R S
Xoo19 R R MR R R R R R S R S
Xoo20 R R R MR R R R R R R S
Xoo21 MR R R R R R R HR R R S
Xoo22 R R R R R R R R R R S
Xoo24 R R R R R R R R R R S
Xoo25 MR R R MR R MS MR HR S R MS
Xoo29 R HR R S R R R R MS R S
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Fig. 3  Photograph depict-
ing the disease response of
RILs to bacterial blight
to produce ­BC1F1 progeny to increase the recipient
parent’s characteristics. The following year, in T.
Aman 2019, B ­ C1F1 plants were again crossed with
the recurrent parent IRRI 154 to create ­BC2F1 prog-
eny, which were named ASRBCR 1077:15, in order
to enhance the recipient parent’s characteristics. For
generation advancement, ­BC2F1 plants were salved
to create ­BC2F2 (ASRBCR 1077:15:7-B) progeny.
Cutting the breeding cycle time is one of the sim-
ple methods for increasing genetic gain (Atlin et al.
2017). To shorten the reproductive cycle for rice, off-
season progeny promotion is required. Rapid Gen-
eration Advance (RGA) is a breeding technique in
which segregating populations are developed at closer
spacing, high temperatures, and short days to reduce
growth duration, making it feasible for several gener-
ations to occur each year. It usually takes four years to
get from F­ 2 to F
­ 5, but with RGA it might just take two
years. Goulden (1939) proposed that hybridization-
derived segregating generations could be rapidly pro-
gressed without selection and that one or two progeny
per plant could be chosen at random in successive
generations. By collecting a single panicle from each
of the 3,000 to 4,000 plants in a cross, we were able
to guarantee that each plant’s offspring was repre-
sented. In the third year (T. Aman 2020), the same
procedure as described above was used to collect one
panicle per plant, and they were kept for the line stage
trial (LST) in the next Boro 2020–21 season.
Following ­F5 generations, LST was carried out
with almost all of the plants being homozygous.
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Progeny rows will now be chosen according to the
ideal characteristics we are looking for, includ-
ing homogeneity, growth length, grain type, disease
resistance, and phenotypic acceptability. In Boro
2020–21, a total of 392 lines consisting of ASRBCR
1077-B-B RGA-B RGA-B RGA (192 lines) and
ASRBCR 1077:15:7-B-B RGA-B RGA (200 lines)
were tested in LST, where we selected 60 lines (48
lines from ASRBCR 1077-B-B RGA-B RGA-B RGA
and 12 lines from ASRBCR 1077:15:7-B-B RGA-B
RGA).
After identifying the three BLB resistance genes
(xa5, xa13, and Xa21) for pyramiding, gene-specific
markers were used for efficient foreground selection
in a total of 392 RILs throughout two generations
­(F5 and ­BC2F4). The xa5 gene was marked with the
marker M440, the xa13 gene with M478Lm, and the
Xa21 gene with M486Lm. Bands of 198 bp, 500 bp,
and 980 bp were seen in PCR results for the xa5,
xa13, and Xa21 genes, respectively (Kumar et al.
2013; Xu et al. 2012), and IRBB66, a resistance
check, verified these findings (Figs. 4, 5, and 6).
Out of a total of 392 lines, 319 had at least one of
the targeted BLB resistance genes, while the other
13 and 60 had at least two or three BLB resistance
genes, respectively (Figs. 4, 5, and 6). In accordance
with the findings of Dokku et al. (2013), these recom-
binant lines demonstrated a high degree of resistance
against fifteen virulent BLB isolates. The efficacy of
a combination of two or more genes is better than
that of a single gene in battling concurrent pathogen
 Euphytica
(2024) 220:60

Table 6  Disease score of 9 advance lines with 2 check varieties

Designation ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR ASRBCR IRBB66 IRRI 154
Isolates 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077-B-B 1077:15:7-B
RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B RGA-B
RGA-B RGA- RGA-B RGA- RGA-B RGA- RGA-B RGA-B RGA-B RGA-B RGA-B RGA-70-1
105-1 141-2 15-1 RGA-182-3 RGA-184-2 RGA-24-1 RGA-63-3 RGA-7-2

Xoo1 2.33 1.87 1.67 7.58 1.5 4.67 1.75 2.3 4.25 1.75 9.33
Xoo5 2.47 2.2 2.5 2.5 3.67 3.83 2.7 0.33 1 3.2 12.92
Xoo7 1.97 3.33 6.67 1.22 2.05 11.68 1.87 1.97 5.17 3.15 13.83
Xoo9 1.33 2.7 1 1.47 2.17 1.67 3 1.77 1.67 1.17 12.83
Xoo15 1 0.35 1.17 3.88 2 1.33 1.5 1 3 1.5 3.5
Xoo16 1.83 1.5 3.5 2.17 2 1 3.67 0.92 2.33 1.67 11.97
Xoo17 7.92 1.47 10.02 6.68 2.5 7.15 3.03 3.33 3.5 2.17 17.58
Xoo18 0.97 1.88 1.53 1.88 2 2.38 1.22 1.43 1.38 2.17 12.55
Xoo19 0.8 2.89 5.97 2.07 2.05 2.5 0.9 1.25 11.82 0.8 11.72
Xoo20 1.83 0.97 3.67 3.8 2.83 1.35 1.82 1.38 1 1 10.38
Xoo21 4.52 2 1.57 1.77 2.13 1.58 1.17 0.17 1.3 1.85 11.1
Xoo22 2.67 1.95 2.17 1.2 2.4 1.77 1.67 0.93 0.83 1.25 10.47
Xoo24 0.87 1.35 2.3 1 1.18 1.8 1.45 0.87 1.47 0.68 11.33
Xoo25 4.85 0.85 3.68 4.77 1.82 8.68 3.82 0.39 11.57 1.73 9.3
Xoo29 2.3 0.47 10.93 10.4 1.43 1.72 1.05 0.72 7.07 0.73 1.33
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Fig. 4  Amplification of
resistant xa5 (158 bp)
gene from RILs; Lane 1:
1000 bp DNA ladder (Pro-
mega, USA), Lane 2: donor
parent (positive control),
Lane 3: recipient parent and
Lane 4–18 advance lines

Fig. 5  Amplification of
resistant xa13 (450 bp)
gene from RILs; Lane 1:
1000 bp DNA ladder (Pro-
mega, USA), Lane 2: posi-
tive control (donor parent),
Lane 3: recipient parent
(negative control) and Lane
4–18 advance lines

Fig. 6  Amplification of
resistant Xa21 (980 bp)
gene from RILs; Lane 1:
1000 bp DNA ladder (Pro-
mega, USA), Lane 2: posi-
tive control (donor parent),
Lane 3: recipient parent
(negative control) and Lane
4–18 advance lines

alterations for virulence, as shown by Mundt (2018),
making the assembly of multiple resistance genes into
a host plant a viable and useful strategy.
In T. Aman 2021, nine RILs were selected
from sixty RILs based on their PAcp, plant height,
growth duration and yield performance. Given the
wide range in plant height, effective tiller, panicle
length, and grain weight among them, these pyra-
mided RILs offer a lot of room for expansion and
growth in terms of their production capacity. The
­ 7 and B
field test of the F ­ C2F6 generation’s improved
pyramided lines showed that the chosen lines had
comparable yield and quality features, as well as
comparable pyramided genes for BLB. Combining
phenotypic and genotypic selection has helped dif-
ferent lines become more resistant to the BLB dis-
ease without lowering yields. These nine RILs were
again trialed at the T. Aman 2022 season with both
parents, which were used as check. Therefore, we
found two best lines that were superior to the check,
and they showed highly resistance against the bacte-
rial leaf blight.

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In a greenhouse, the pathogenicity was tested with
virulent strains of X. oryzae pv. oryzae that caused
leaf blight on the surface of rice plants two weeks
after they were artificially infected (Fig. 2). Bacte-
rial suspension was clip-inoculated onto 45-day-old
plants of several advance lines. After 15 days of inoc-
ulation, symptoms appeared as water-soaked lesions
that extended wavy-like from the margin of the leaf
blade and attacked leaf sheaths and culms. The exper-
imental area bordering the healthy part of the plant
displayed water-soaked blighted lesions that swiftly
spread to cover significant areas of the leaf blade,
turned yellowish-white, and later became greyish.
These symptoms were not seen in the control plants.
The re-isolated bacteria were similar to the initial
Xanthomonas oryzae pv. oryzae cultures. The disease
reaction of the plant was measured by the length of
the lesion, and these results back up those of Horino
et al. (1983), who did a pathogenicity test to describe
a large number of X. oryzae pv. oryzae isolates. In
some cases, responses were simply categorized as
resistant (5.0 cm) and susceptible (5.1 cm).
In our bioassay study, we found that nine pyra-
mided lines with at least two BLB resistance genes
were very resistant (Table 5). Three pyramided
lines (ASRBCR 1077-B-B RGA-B RGA-B RGA-
7-2, ASRBCR 1077-B-B RGA-B RGA-141-2, and
ASRBCR 1077-B-B RGA-B RGA-184-2) contain-
ing xa5 + xa13 + Xa21 outperformed the RILs with
other gene combinations in terms of resistance
(Dash et al. 2016; Finatto et al. 2015; Mew 1987;
Zhai and Zhu 1999). Similar to the findings of prior
investigations (Das and Rao 2015; Dokku et al.
2013; Pradhan et al. 2015; Suh et al. 2013), these
3 RILs each had an average of 1.25 cm, 1.72 cm,
and 2.16 cm of lesion length infected with the fif-
teen BLB races (Table 6). This happens when two
or more genes work together instead of just one.
This is called synergistic action or quantitative
complementation (Sanchez et al. 2000). On the
basis of lesion length, ASRBCR 1077-B-B RGA-B
RGA-B RGA-105-1 was resistant to twelve isolates,
moderately resistant to two isolates and moder-
ately sensitive to one isolate. Once again, ASRBCR
1077-B-B RGA-B RGA-B RGA-141-2 was highly
resistant to two isolates, resistant to twelve iso-
lates and moderately resistant to one isolate. Also,
ASRBCR 1077-B-B RGA-B RGA-B RGA-15-1
was resistant to eleven isolates, moderately resistant
Page 13 of 15 60
to two isolates, moderately sensitive to one isolate
and sensitive to one isolate. In addition, ASRBCR
1077-B-B RGA-B RGA-B RGA-182-3 was resistant
to nine isolates, moderately resistant to three iso-
lates, moderately sensitive to two isolates, sensitive
to one isolate. Furthermore, ASRBCR 1077-B-B
RGA-B RGA-B RGA-184-2 was resistant to four-
teen isolates and moderately resistant to one isolate.
In addition, ASRBCR 1077-B-B RGA-B RGA-B
RGA-24-1 was resistant to ten isolates, moderately
resistant to two isolates, moderately sensitive to two
isolates, sensitive to one. Additionally, ASRBCR
1077-B-B RGA-B RGA-B RGA-63-3 was resistant
to twelve isolates and moderately resistant to three
isolates. Furthermore, ASRBCR 1077-B-B RGA-B
RGA-B RGA-7-2 was highly resistant to three iso-
lates, resistant to eleven isolates and moderately
resistant to one isolate. In addition, ASRBCR
1077:15:7-B RGA-B RGA-70-1 was resistant to
nine isolates, moderately resistant to one isolate,
moderately sensitive to three isolates and sensitive
to two. In addition, IRBB66 was resistant to thirteen
isolates and moderately resistant to two isolates
(Table 5). In addition, IRRI 154 was moderately
resistant to one isolate, moderately sensitive to two
isolates, sensitive to eleven isolates and highly sen-
sitive to one isolate.
Figure 3 and Table 6 illustrate the disease
responses of advanced lines. The length of lesions
varied between 0.17 cm and 17.58 cm across all
genotypes. ASRBCR 1077-B-B RGA-B RGA-7-2
had the shortest lesion length compared to the resist-
ant check strain IRBB66 (0.17 cm) and is considered
resistant. The line with the longest lesion was IRRI
154 (17.58 cm). The remaining advance lines exhib-
ited either moderate resistance or susceptibility to fif-
teen isolates of Xoo.
Acknowledgements The authors would like to express their
gratitude to Dr. F H Ansarey, President, ACI Limited, Dhaka
1212, Bangladesh, for providing continual emotional support,
and assistance with lab equipment.
Author’s contributions All authors contributed to the study
conception and design. Material preparation, data collec-
tion and analysis were performed by Md. Ariful Islam, Md.
Moniruzzaman Hasan and Tanbin Akter. The first draft of the
manuscript was written by Md. Ariful Islam and Md. Moniruz-
zaman Hasan and all authors commented on previous versions
of the manuscript. All authors read and approved the final
manuscript.
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 60 Page 14 of 15
Funding The authors declare that no funds, grants, or
other support were received during the preparation of this
manuscript.
Data availability The datasets generated during and/or ana-
lysed during the current study are available from the corre-
sponding author on reasonable request.
Declarations
Competing interests The authors declare that they have no
conflict of interest regarding this manuscript and research.
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