J Intl Cooper Agric Dev 2020; 18: 18–28

Journal ofInternational Cooperation for
Agricultural Development

Original

Evaluation of Backcrossed Pyramiding
Lines of the Yield-related Gene andthe Bacterial Leaf Blight Resistant Genes
Shuto Yamada1), Yusuke Kurokawa1), Keisuke Nagai1), Rosalyn B Angeles-Shim1, 5),
Hideshi Yasui2), Naruto Furuya3), Atsushi Yoshimura2), Kazuyuki Doi4),
Motoyuki Ashikari1), and Hidehiko Sunohara1, 6)
1) Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku Furo-cho, Nagoya, 464-8601 Japan

2) Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Nishi-ku Motooka, Fukuoka, 812-8581 Japan

3) Laboratory of Plant Pathology, Faculty of Agriculture, Kyushu University, Nishi-ku Motooka, Fukuoka, 812-8581 Japan
4) Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku Furo-cho, Nagoya, 464-8601 Japan
5) Current address: Department of Plant and Soil Science, Texas Tech University, Lubbock, Texas, USA 79416
6) Current address: Faculty of Advanced Science and Technology, Kumamoto University, Chuo-ku Kurokami,

Kumamoto, 860-8555 Japan

Received: September 12, 2019 Accepted: December 30, 2019
Abstract. The yield-related gene, WFP increases grain number by increasing the primary branch number per panicle in rice.
In the present study, WFP was introgressed from ST-12 to IRBB60, a pyramiding line having the genes Xa4, xa5, xa13 andXa21 for BLB resistance in the IR24 genetic background. The pyramided lines PL-1, PL-4 and PL-5 that were included in theinitial BC2F2 selections based on their improved PBN were selected for further agronomic evaluation and generation advance
up to BC2F3. All three lines recorded significantly higher PBN and GN due WFP introgression, although the loss of at least
two BLB genes during the breeding process resulted in the variable response of the lines to the different BLB races. Amongthe lines, only PL-5 showed a significantly higher estimate of actual yield measured in terms of panicle weight per square
meter compared to IRBB60. PL-5 also exhibited resistance to five out of the six BLB races used for resistance screening.
Despite the negative effects in grain size, the improved yield estimates, as well as the validated BLB resistance of PL-5 makes
it a suitable candidate for cultivar adoption under the tropical rice ecosystem of Southeast Asia.
Key words: Rice, Wealthy Farmer’s Panicle (WFP), bacterial leaf blight (BLB), pyramiding, high-yielding

Introduction
By 2050, the current world population is expected toreach 9.7 billion (https://www.un.org/development/desa/en/). An increase of this magnitude poses a very serious
threat to the current state of global food security (http://
www.fao.org/home/en/). To feed 9 billion people, global
cereal production alone needs to increase by 38% in the
Corresponding author: Hidehiko Sunohara, e-mail: hsunohara@
kumamoto-u.ac.jp
18 J Intl Cooper Agric Dev 2020next 34 years1).
Rice, wheat and maize constitute approximately
50% of the calorie source of the human diet2). Amongthese three, rice serves as a staple for more than half of
the world’s population. Although rice is cultivated in morethan 100 countries across the globe, more than 90% of
rice is still produced and consumed in Asia (GRiSP, http://
www.grisp.net/main/summary). In recent years, a drastic
increase in demands for rice has also been reported in
Africa3) (GRiSP, http://www.grisp.net/main/summary).
Given the importance of rice as a staple food, increasing

rice production would play a critical role in securing world
food supply.
Marker-assisted selection (MAS) is powerful tool
for an efficient breeding. Since MAS makes it possible
to distinguish plants into which target genes/QTLs havebeen introgressed during nursery, field area and efforts forindividual selection of breeding reduce. Also, target genes/
QTLs can be introgressed more reliably than visually
selection. MAS has been successfully used in breeding
programs to improve agronomic traits, as well as biotic
and abiotic stress resistance in rice. In the past decades,
MAS have significantly shortened the time requirement
while increasing the precision and efficacy by whichtarget genes/QTLs are transferred across different genetic
backgrounds4, 5). To date, several rice cultivars have been
improved through MAS and these include the improved
varieties developed under the Wonder Rice Initiativefor Food Security and Health (WISH) project (http://motoashikari-lab.com/wp/wp-content/uploads/2018/11/
WISH_catalogue_lite4.pdf).
To effectively utilize MAS in breeding programs,
genes/QTLs underlying important traits need to be mappedand identified. In rice, many genes/QTLs that are related
to the sink ability have been identified including GN1a/OsCKX2 6), WFP/IPA17, 8), NAL1/SPIKE/GPS 9, 10, 11),
APO1/SCM2 12, 13, 14), qSW2 15), GW2 16), GW6a17) andGS3 18, 19). Conversely, several genes/QTLs that are related
to the source or translocation ability of rice has also beenreported20, 21, 22) that includes AMY2A23), BAM223) and
GWD124).
Wealthy Farmer’s Panicle (WFP)/Ideal Plant
Architecture 1 (IPA1) is a yield-related gene that encodes
the plant-specific transcription factor OsSPL147, 8). OsSPL14
contains a microRNA-targeted sequence in an exon that istargeted and cleaved by OsmiR156. OsSPL14 functions in
the regulation of primary branch number and is therefore
associated with grain number. The WFP allele from ST-12
has been reported to increase grain number per panicle inrice7). Advanced breeding lines of the japonica rice cultivar
Nipponbare and several other indica cultivars having the
WFP allele from ST-12 showed at least 28% increase in
grain yield compared to the original varieties7, 25).
Bacterial leaf blight (BLB) caused by Xanthomonas
oryzae pv. oryzae (Xoo) is one of the most serious diseasesof rice throughout the world. In some regions in Asia,
BLB can cause yield losses of up to 50%26). The mosteffective approach to combat BLB is the use of resistantcultivars27). To date, many resistant genes against BLB
have been identified. Huang et al. focused on four BLB
resistance genes/QTLs namely Xa4, xa5, xa13 and Xa21,
and pyramided all four resistance genes in the IR24 genetic
background to generate IRBB6028). IRBB60 has resistanceto six races of Xoo from the Philippines28). Currently, theBLB genes xa5, xa13 and Xa21 have been identified andcharacterized29, 30, 31).
In this study, we aimed to pyramid WFP from
ST-12 to IRBB60, a rice cultivar having four genes for
BLB resistance. The resulting pyramided lines were
evaluated for yield and yield components, as well as fortheir resistance to BLB races from Vietnam, Myanmar
and India. Results of the preliminary assessment of theagronomic performance of advanced backcrosses lines
identified PL-5, a pyramiding line with higher yieldcompared and comparative BLB resistance relative to therecipient parent IRBB60.

Materials and Methods
Marker-assisted introgression of WFP in the pyramiding
line IRBB60

IRBB60, IR24, ST-12 and three pyramiding lines(PL-1, PL-4 and PL-5) at the BC2F4 generation wereused in the study. IRBB60 is a pyramiding line having
the four bacterial leaf blight (BLB) resistant genes, Xa4,
xa5, xa13 and Xa21 in the genetic background of IR2428),
whereas ST-12 is the donor of the yield-related gene,
WFP7). Phenotype and marker-assisted backcrossingof the initial hybrids generated from crosses between
IRBB60 and ST-12 was carried out up to the BC2F4
generation (Fig. 1). An SSR marker (forward primer:
GCGGTAACAAACCAACCAACC, reverse primer:
AAAGCAGGACACAGTCACACAGG)
wasused
to monitor the introgression of WFP in the IRBB60
background. In 2016, six BC2F3 lines (PL-1, PL-2, PL4, PL-5, PL-6 and PL-7) were selected for preliminary
evaluation of yield and yield components in the field.
Based on this preliminary investigation, three pyramiding
lines namely PL-1, PL-4 and PL-5 were selected andadvanced for further performance evaluation in the field.
All test materials were raised in the greenhouse forfour weeks before transplanting them in a rice paddy at
the Togo Field Center for Research and Education of
Nagoya University in Aichi, Japan (35º06' north, 137º04'
east) in 2017. The seedlings were transplanted separately
in 3 rows (1.2 m) at a density of 22.2 plants/m2 and at aplant spacing of 30cm x 15cm. All materials were grownin three replications in randomized blocks and under low
fertilizer conditions i.e. 300 kg K-P-N (16-12-14)/ha only
before transplanting.
Evaluation of the pyramiding lines for agronomic traits

Before sampling panicles from all materials, data on
the days to heading (DH), plant height (PH) and panicle
length (PL) of five plants per block were recorded fromJ Intl Cooper Agric Dev 2020 19

Fig. 1. Process of developing the pyramiding lines. X mark in a circle means selfing. The introgressionof WFP from ST-12 was confirmed by MAS in BC1F1, BC2F1 and BC2F3.

plant stands in the paddy. At 40-45 days after heading, allpanicles from five plants from each block were sampled.
After sampling, panicle weight per plant (PW), panicle
number per plant (PN), primary branch number per main
panicle (PBN) and grain number per main panicle (GN)were measured. To estimate the actual yield of eachpyramiding line in the field, panicle weight per plant wasmeasured and converted to panicle weight per square
meter (PWm2). Only plants within rows were sampled to
avoid border effects. All panicles were dried for a week in
an incubator set at 42°C. To determine fertility ratio (FR),the three largest panicles including the main panicle werechosen from each plant. FR was calculated by dividing thenumber of filled spikelets by the total number of spikelets,
multiplied by 100. The grain weight and number of filled
spikelets of the three largest panicles were measured andconverted to 1000-grains weight per main three panicles(TGW). Grain shape based on grain length (GL), width(GW) and thickness (GT) was determined from ten
randomly selected spikelets in each plant. For the statisticalanalyses, plants showing maximum and minimum PW ineach plot were excluded.

Evaluation of the pyramided lines for bacterial leaf
blight resistanceSix races of Xanthomonas oryzae pv. oryzae (Xoo)causing bacterial leaf blight (BLB) from Vietnam(HAU01043, HAU0209, HAU02021-2 and HAU020246)32), Myanmar (HKM13)33) and India (IND2-3) were used
to evaluate the resistance of the experimental materials tothe pathogen. At booting stage, the plants were inoculatedwith Xoo using the leaf clipping method34). A preliminary
test was conducted using IR24 to confirm the pathogenicity
and virulence of the six BLB races. Immediately after
that, all the experimental materials including IR24 as thesusceptible control and IRBB60 as the resistant control,
were screened against the pathogen. After two weeks
from inoculation, lesion lengths were measured, and theresistance/susceptibility of each material was evaluated.
Classification of the response of materials as resistant,moderate, susceptible or partially resistant was carried out
based on a comparison to the response of the susceptiblereference IR24 and the resistant reference IRBB60 using
Tukey’s HSD (Table 1). Screening for BLB resistanceincluding maintenance and culture of the pathogen was

Table 1. Classification of the response of materials to the BLB races based on acomparison to the response of the susceptible reference IR24 (S) andthe resistant reference IRBB60 (R).
Statistical difference toIR24 (S)Statistical difference to
IRBB60 (R)Significant*Significant
Not significant
Not significant

Not significant
Significant
Significant
Not significant

Response
ResistantModerate
Susceptible
Partially resistant***; significant according to Tukey’s HSD (p<0.05).
**; only in the case that evaluation value is between the values of the references.
20 J Intl Cooper Agric Dev 2020

carried out in Laboratory of Plant Pathology, Faculty ofAgriculture, Kyushu University.
Genotyping-by-sequencing analysis of the pyramiding
lines

Genotyping-by-sequencing (GBS) was conducted forthe three pyramiding lines at the BC2F4 generation using
the Illumina Miseq sequencing system and Illumina Miseq
Reagent Kit v3 (150 cycles)35, 36). Three plants from eachof the pyramiding line, as well as IRBB60 and ST-12 were
genotyped by GBS for whole genome. After sequencing,
the reads were trimmed, and genotypes were called andfiltered using TASSEL-GBS37). SNPs with minor allele
frequency (MAF) of >0.02, as well as lines with >0.2 lost
genotype data and with >0.125 degree of heterozygous
sites were removed from genotyping. Finally, imputationand error correction were carried out with FSFHap in
TASSEL 4.038).
Statistical analysis

T-test and Tukey’s HSD were performed using thefree statistical software R (https://www.r-project.org/).
A significant difference in Tukey’s HSD was set at p<0.05.
R was also used for the randomization of blocks in thefield design.

ResultPhenotypic evaluation of the three pyramiding lines
for yield components

The pyramiding lines PL-1, PL-2, PL-4, PL-5, PL-6 andPL-7 in the genetic background of IRBB60 were selectedvisually from a BC2F2 population for their high primarybranching number. The introgression of WFP from ST-12
in these six lines at the BC2F3 generation was confirmed
by MAS. Evaluation for agronomic and yield-related traits
showed that except for PL-6, all the pyramiding lines had

significantly more PBN than IRBB60 (Table 2). PL-6 andPL-7 recorded significant lower PN (panicle number per
plant) than IRBB60. The reduction of PN is the negative
effect for actual yield, hence both PL-6 and PL-7 were
removed from the candidate lines. Similarly, PL-2 was removed from the candidate lines because of comprehensive
gross morphology. Based on the overall agronomic performance and gross morphology of the pyramiding lines,
we selected PL-1, PL-4 and PL-5 for further generation
advance and agronomic evaluation (Fig. 2).
At the BC2F4 generation, PL-1, PL-4 and PL-5 were
evaluated for yield components including DH, PH, PL,
PN, GN, PBN, FR, TGW, GL, GW and GT (Table 3).
Compared to IRBB60, PL-1 recorded a significant 4-day
delay in a heading, whereas PL-5 was significantly earlyby 4 days. Heading date in PL-4 was equivalent to that of
IRBB60. PN decreased by 0.33 to 1.33 in all pyramiding
lines compared to IRBB60. GN (grain number per main
panicle) and PBN (primary branch number per main
panicle) of the three pyramiding lines were significantly
higher than those of IRBB60, suggesting the positiveeffects of WFP introgression from ST-12. The PBN of
ST-12 was twice higher than that of IRBB60 and IR24.
FRs (fertility ratio per three main panicles) of IRBB60,
IR24 and PL-5 showed high values, while those of ST-12,
PL-1 and PL-4 were low, below 77%. Among the threepyramiding lines, PL-5 showed the highest FR (92.56 ±
0.40%). The FRs of PL-1 and PL-4 were 76.84 ± 2.37%
and 72.64 ± 0.61% respectively, which were equivalent tothat of ST-12.
In PWm2, PL-5 recorded a significantly higher yield(845.50 ± 57.20 g/m2) than IRBB60 (782.42 ± 47.90g/m2), with the rate of increase in PL-5 reaching up to
8.1% of IRBB60. ST-12, the donor of WFP, had significantly lower PWm2 (712.4 ± 43.5 g/m2) than IRBB60 (Fig.
3). The panicle weight of PL-1 and PL-4 were statisticallyequivalent to that of IRBB60.

Table 2. Yield-components of the pyramiding lines at BC2F3 in the IRBB60 background.
PH (cm)PL (cm)

PN

GN

PBN

IRBB60ST-12

97.00 ± 4.36 ac*
115.00 ± 7.55 bd

26.40 ± 0.51 ab
25.43 ± 1.33 b

11.33 ± 0.58 a
7.00 ± 1.00 bc

203.33 ± 17.79 a
280.67 ± 43.35 ab

12.33 ± 0.58 a
21.00 ± 4.00 bc

PL-1
PL-2
PL-4
PL-5
PL-6
PL-7

119.33 ± 3.21 b
98.50 ± 6.36 ac
114.33 ± 2.52 bd
102.33 ± 5.13 acd
89.33 ± 6.43 a
103.67 ± 1.15 cd

29.26 ± 0.61 a
25.48 ± 1.52 ab
26.40 ± 1.03 ab
23.43 ± 0.99 ab
24.90 ± 1.73 ab
25.30 ± 1.63 b

8.67 ± 0.85 ac
11.00 ± 1.41 ad
11.33 ± 1.53 a
8.33 ± 1.53 ab
5.33 ± 0.58 b
7.67 ± 1.53 bcd

314.67 ± 66.83 ab
281.00 ± 31.11 ab
312.67 ± 33.56 ab
274.33 ± 15.53 ab
204.00 ± 33.05 a
354.33 ± 62.01 b

23.33 ± 1.53 b
23.00 ± 1.41 b
24.67 ± 0.58 b
20.33 ± 1.53 bc
17.00 ± 1.00 ac
21.00 ± 1.00 bc

Numbers represent average ± standard deviation.
PH=plant height; PL=panicle length; PN=panicle number per plant; GN=grain number per main panicle; PBN=primarybranch number per main panicle.
*; different letters indicate significant differences at p<0.05 by Tukey’s HSD.
J Intl Cooper Agric Dev 2020 21

Fig. 2. Gross morphology of the plants and panicles of the experimental materials. A-F: Gross plant morphology at the
ripening stage, G-L: Gross panicle morphology at the ripening stage. A and G: IRBB60, B and H: IR24, C and
I: ST-12, D and J: PL-1, E and K: PL-4 and F and L: PL-5. Bar = 20cm (A–F), 5cm (G–L).

Table 3. Yield-components of the pyramiding lines at BC2F4 in the IRBB60 background.
Line NameIRBB60
IR24
ST-12
PL-1
PL-4
PL-5
Line NameIRBB60
IR24
ST-12
PL-1
PL-4
PL-5

DH (days)

PH (cm)PL (cm)

PN

GN

PBN

105.00 ± 0.50
109.44 ± 0.33**
95.78 ± 0.33**
109.11 ± 0.44**
105.22 ± 0.53
101.11 ± 0.67**

94.67 ± 1.55
83.89 ± 1.38**
101.89 ± 1.14**
103.33 ± 2.39**
96.67 ± 1.98
95.89 ± 1.71

23.89 ± 0.44
22.22 ± 0.58**
23.78 ± 0.53
25.78 ± 1.14*
23.22 ± 0.50
22.00 ± 0.24**

10.44 ± 0.65
10.33 ± 0.80
8.89 ± 0.50**
9.11 ± 0.93
9.44 ± 0.41*
10.11 ± 0.80

160.22 ± 9.47
161.33 ± 10.07
283.11 ± 15.61**
219.89 ± 16.68**
231.78 ± 10.14**
239.89 ± 9.29**

12.00 ± 0.37
12.78 ± 0.37*
22.33 ± 0.96**
20.11 ± 0.65**
21.44 ± 1.40**
20.56 ± 0.75**

FR (%)TGW (g)

GL (mm)

GW (mm)GT (mm)94.90 ± 0.26
88.14 ± 0.79**
72.75 ± 1.14**
76.84 ± 2.37**
72.64 ± 0.61**
92.56 ± 0.40**

26.45 ± 0.08
25.65 ± 0.11**
23.73 ± 0.11**
27.55 ± 0.40**
24.40 ± 0.09**
20.83 ± 0.11**

9.46 ± 0.02
9.11 ± 0.03**
9.87 ± 0.02**
9.56 ± 0.06**
9.47 ± 0.02
8.60 ± 0.01**

2.41 ± 0.01
2.52 ± 0.01**
2.33 ± 0.01**
2.45 ± 0.02**
2.41 ± 0.01
2.22 ± 0.01**

1.96 ± 0.01
1.97 ± 0.01*
1.94 ± 0.01**
1.91 ± 0.02**
1.86 ± 0.01**
1.79 ± 0.01**

Numbers represent average ± standard error.
DH=days to heading; PH=plant height; PL=panicle length; PN=panicle number per plant; GN=grain number per main panicle;
PBN=primary branch number per main panicle; FR=fertility ratio per three largest panicles in a plant; TGW=1000-grain weight per
three largest panicles in a plant; GL=grain length; GW=grain width; GT=grain thickness.
*, **; indicate significant difference from IRBB60 at p<0.05 and p<0.01 by t-test, respectively.

Based on these results, the high yield of PL-5 can be
attributed to the increase in GN and PBN. In spite of PL-5
having 49.7% higher GN than IRBB60, PWm2 of PL-5 was
only 8.1% higher. As a major cause of this phenomenon,
we focused on grain size. The grain size is expected toaffect TGW (1000-grains weight per main three panicles).
Compared to the TGW of IRBB60 (26.45 ± 0.08 g), theTGW of PL-5 (20.83 ± 0.11 g) was significantly lower(Table 3). Similarly, PL-5 recorded smaller grains in termsof GL (8.60 ± 0.01 mm), GW (2.22 ± 0.01 mm) and GT(1.79 ± 0.01 mm) compared to those of IRBB60 (9.46 ±22 J Intl Cooper Agric Dev 2020

0.02 mm, 2.41 ± 0.01 mm, 1.96 ± 0.01 mm) (Table 3). The
smaller grains of PL-5 negatively affected its yield.
Evaluation of the pyramiding line for resistance tobacterial leaf blight

The response of the experimental materials whenchallenged with BLB races from Vietnam (HAU01043,
HAU0209, HAU02021-2 and HAU02024-6), Myanmar(HKM13) and India (IND2-3) are shown in Fig. 4.
IRBB60 showed strong resistance to the six BLB races,with lesions lengths ranging from 0 to 1.2 cm. In contrast,Confirmation of introgression of targeted genes
with genotyping-by-sequencing

GBS analysis validated the introduction of the WFP
allele from ST-12 and the segregation of the four BLB
resistant genes of IRBB60 in each of the pyramiding
lines at BC2F3 (Table 4). All pyramiding lines carry the
Xa21 allele from IRBB60 and the xa13 allele from ST-12.
PL-1 and PL-4 have the Xa4 allele from IRBB60 but not
PL-5 which has the Xa4 from ST-12. The xa5 allele from
IRBB60 was introgressed in the homozygous form in PL-5
but occurs in a heterozygous form in PL-4. PL-1 has the
ST-12 allele for xa5.

Discussion

Fig. 3. Panicle weight per square meter (PWm2) of theexperimental materials. * and ** indicate significant
difference from IRBB60 at p<0.05 and p<0.01, respectively, by t-test. Error bars show standard error.

IR24 and ST-12 were susceptible to the six BLB races,with lesions lengths ranging from 13.3 to 25.2 cm in IR24
and from 10.5 to 26.8 cm in ST-12. The pyramiding lines
PL-1, PL-4 and PL-5 showed differential responses tothe six BLB races. PL-1 exhibited resistance to the five
BLB races from Vietnam (HAU0209, HAU02021-2 andHAU02024-6), Myanmar (HKM13) and India (IND2-3),
and moderate to HAU01043. PL-4 exhibited resistanceto HAU0209, moderate to IND2-3, partially resistance toHKM13 and susceptibility to HAU01043, HAU020212 and HAU02024-6. PL-5 exhibited resistance to thefive BLB races from Vietnam (HAU01043, HAU0209,
HAU02021-2 and HAU02024-6) and India (IND2-3), andpartially resistance to HKM13 from Myanmar.

The pyramiding lines (BC2F4) reported in the present
study were developed from the cross between ST-12 (usedas the donor of WFP) and IRBB60 (used as the recipientparent with BLB resistance). Introgression of WFP from
ST-12 in the PLs was confirmed by MAS and GBS (Fig.
1, Table 4). As a result, we developed PL-5, a pyramiding
line with higher yield compared to the recipient parent,
IRBB60 (Fig. 3). The high yield of PL-5 can be attributed
to the increase in both GN and PBN (Table 2, Table 3)due to the introgression of WFP from ST-12. Miura et al.
reported that the introgression of WFP from ST-12 cansignificantly increase both GN and PBN in lines having thegenetic background of the japonica rice cultivar Nipponbare7). Similarly, introgression of WFP has been reported
to increase GN and PBN of several rice cultivars withindica genetic backgrounds25). In the present study, thepositive effects of WFP from ST-12 was also observed in
the genetic background of IRBB60, suggesting that WFP
expression can increase GN and PBN in both japonica andindica rice backgrounds, and that WFP from ST-12 is a
useful genetic resource that can be used to significantly
improve the yield of rice.
PL-5 recorded 49.73% higher GN and 71.33% higher
PBN compared to IRBB60. Despite the significant increasein GN and PBN, PL-5 recorded a remarkably low increase

Table 4. Genotypes in targeted genes/QTL in the three pyramiding lines in the previous generation (BC2F3).
Xa4
xa5
xa13
Xa21
WFP

Chromosome

RAP-IDQTL location*

IRBB60

ST-12

PL-1

PL-4

PL-5

Exp**

11
5
8
11
8

Os05g0107700
Os08g0535200
Os11g0559200
Os08g0509600

Around 28.5Mb
-

A
A
A
A
A

B
B
B
B
B

A
B
B
A
B

A
H
B
A
B

B
A
B
A
B

A
A
A
A
B

*; physical distance showed in RAP-db site (https://rapdb.dna.affrc.go.jp/).
**; expected genotypes.

J Intl Cooper Agric Dev 2020 23

Fig. 4. The response of the experimental materials to the six races of Xoo. A: Lesion length on leaves of the experimental
materials. Different letters indicate significance at p<0.05 by Tukey’s HSD. Error bars show standard error. B: Lesions in leaves infected by the six BLB races. The reaction of the experimental materials to the BLB races is shownin two leaves each. The order of BLB races in each panel is HAU01043, HAU0209, HAU02021-2, HAU02024-6,
HKM13 and IND2-3 from left to right. The inoculation site corresponds to the top of each panel. Bars = 5cm.

24 J Intl Cooper Agric Dev 2020

of 8.06% in yield (PWm2) compared to IRBB60. A similar
phenomenon was observed in PL-1 and PL-4. Rice yieldhas four important components namely PN, GN, FR and
TGW. In PL-5, the main negative factor was TGW, which
was 21.25% lower than IRBB60. Based on the results ofthe current study, an improvement in TGW can further
increase rice yield. In PL-1, the main negative factors were
PN and FR which were 12.74% and 19.03% lower than
IRBB60, respectively. In PL-4, the negative factors were
PN, FR and TGW which were 9.58%, 23.46% and 7.75%
lower than IRBB60, respectively.
The low TGW of PL-5 may be unrelated to the functionof WFP. PL-5 has smaller grains, giving it lower TGW
than IRBB60. The donor parent ST-12 do not have small
grains, and WFP/IPA1 from ST-12 has not been reported to
be involved in the regulation of grain size. We hypothesizethat the genetic background of PL-5 which resulted fromrandom recombination between the ST-12 and IRBB60
genome contributed to its smaller grains.
Miura et al. suggested that WFP from ST-12 is involvedin the regulation of shoot branching in the vegetative
stage7). It was also suggested that WFP/IPA1 from thejaponica cultivar Aikawa 1 and from the japonica cultivar
Shaoniejing reduces tiller number7, 8). While the presenceof WFP/IPA1 allele may increase GN, it may have negative effects on PN. In the present study, an increase in GN
due to WFP/IPA1 introgression resulted in a concomitantdecrease in PN. To suppress any reduction in PN due to
WFP introgression, it is necessary to modify factor(s) related to controlling PN specifically in the vegetative stage.
It is considered that the low FR of PL-1 and PL-4 occurred as a result of the inability of the source to keep up
with the improved sink ability of both lines as conferred
by WFP. In PL-5, a remarkable reduction in grain size has
been confirmed that allowed the source to cope with theincrease in sink. In the context of sink-source relationship, WFP is a sink ability-related gene. Improvement of
the sink is necessary to improve yield, but this requires
a concomitant improvement in the source39). Efficient
breeding for high yield in rice requires elucidation of
the genetic factors regulating the ability of the source togenerate photosynthetic assimilates that will fill the sink.
Improving the photosynthetic ability of plant tissues withsource functions may be the key to breaking through theyield potential of the current high-yield varieties40).
The present study aimed to pyramid WFP with four BLB
resistant genes already present in the IRBB60 background.
The three selected pyramiding lines however, had WFP
and at least two BLB resistant genes each (Fig. 4, Table4). The incomplete maintenance of all four BLB resistancegenes in the pyramiding line accounts for the variability inthe response of each line to each of the BLB races. While

the BLB resistant genes are not linked, xa13 is linked to
WFP at approximately 1.5 Mb distance downstream. In theprocess of developing these pyramiding lines, genotyping
was carried out to monitor the introgression of only WFP.
To ensure that all four BLB resistant genes are maintainedin the pyramided line, these genes should also be monitored in the backcrossed and selfed generations by MAS.
Despite losing at least two BLB resistant genes, PL-5
remains resistant to four BLB races from Vietnam and arace from India, whereas PL-1 is resistant to three racesfrom Vietnam and races from Myanmar and India. These
results indicate the both PL-1 and PL-5 can be cultivatedin areas with a reported incidence of the specific BLB infection. Overall, the significantly improved yield of PL-5
in combination with its resistance to five known BLB racesfrom Vietnam and India makes this pyramiding line a viable candidate for high yield line with adoption in tropicalrice ecosystems in Southeast Asia.
Acknowledgement
This research was funded by the Japan International
Cooperation Agency (JICA) as part of the Wonder Rice
Initiative for Food Security and Health (WISH) Project,
and the Canon Foundation. This research was also supported by Science and Technology Research Partnershipfor Sustainable Development (SATREPS), and Japan Science and Technology Agency (JST, JPMJSA1706) / JICA.
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収量関連遺伝子と白葉枯病抵抗性遺伝子の戻し交配によるピラミディング系統の評価
山田修土 1)， 黒川

裕介 1)，永井啓祐 1)，Rosalyn B Angeles-Shim1, 5)，安井

古屋

成人 3)， 吉村

淳 2)，一行 4)，芦苅土井基行 1)，春原秀 2)，

英彦 1, 6)

1) 名古屋大学生物機能開発利用研究センター
2) 九州大学農学部植物育種学研究室
3) 九州大学農学部植物病理学研究室
4) 名古屋大学大学院生命農学研究科
5) 現所属 : Department of Plant and Soil Science, Texas Tech University, Lubbock, Texas, USA 79416
6) 現所属 : 熊本大学先端科学研究部（理）要旨
イネ収量関連遺伝子 WFP は、一次枝梗数増加による着粒数増加をもたらす。IR24 に白葉枯病抵抗性遺伝子群（Xa4、xa5、
xa13、Xa21）を集積した IRBB60 へ、ST-12 が持つ WFP の導入を行った。IRBB60 を反復親とした戻し交雑後代 BC2F2 とBC2F3 世代で、DNA マーカーの利用と一次枝梗数を含む収量形質の評価から、3 ピラミディング系統（PL-1、PL-4、PL-5）を選抜した。BC2F4 において、これらは WFP による一次枝梗数と着粒数の増加を示したが、4 白葉枯病抵抗性遺伝子の集積が不完全で、白葉枯病レース群への反応の差が見られた。しかし、PL-5 は IRBB60 よりも収量増加が見込まれ、今回供試した白葉枯病レース（ベトナム、ミャンマー、インド由来 6 レース）の内 5 レースに対して抵抗性を示したことから、PL-5
は東南アジアの熱帯地域での展開を見込んだ候補系統となり得ると考えられた。

キーワード：イネ、WFP 遺伝子、白葉枯病抵抗性、ピラミディング、高収量性

28 J Intl Cooper Agric Dev 2020