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International Journal of Agricultural

Science and Research (IJASR)


ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 5, Oct 2016, 339-346
TJPRC Pvt. Ltd

IMPROVING BLAST RESISTANCE IN PARENTAL LINE OF RICE


HYBRID THROUGH MARKER ASSISTED SELECTION
T. SHALINI, P. GOVINTHARAJ, M. AMEENAL, S. MANONMANI & S. ROBIN
Department of Rice, Centre for Plant Breeding and Genetics, Tamil Nadu, India
Agricultural University, Coimbatore, Tamil Nadu, India
ABSTRACT
The present investigation was carried out to introgress blast resistance gene (Piz) into restorer line of popular
rice hybrid CO 4 (COMS 23A / CB 174 R) by Marker Assisted Breeding. Zenith was used as donor for blast resistance
(Piz). Foreground selection was done at F1 and F2 generation using RM 549 SSR marker. Seven plants having the Piz
resistance gene for blast in heterozygous condition were selected in F1 generation. Molecular analysis in F2 generation of
CB 174 R x Zenith identified blast resistance gene in twenty three plants out of 36 plants genotyped. Most of the plants
which were positive were also resistant phenotypically. Chi square analysis confirmed the 3:1 ratio indicating monogenic
control of resistance in the cross. F3 lines of CB 174R x Zenith were phenotypically screened for rice blast under natural
comparison to the susceptible control CO 39 (score of 9).
KEYWORDS: Blast, Screening, Rice, MAS

Received: Sep 13, 2016; Accepted: Oct 07, 2016; Published: Oct 13, 2016; Paper Id.: IJASROCT201641

INTRODUCTION

Original Article

conditions at Gudalur. Among eight lines, two lines of CB 174R x Zenith, were resistant to leaf blast (score 3) in

Rice (Oryza sativa L.), a member of the family Poaceae is widely grown in tropical and subtropical
regions (Ezuka and Kaku, 2000). It plays a major role as it is the staple food for over 2.7 billion people worldwide.
Rice blast being the devastating disease, is an important limiting factor for rice cultivation world-wide which
causes annual losses of up to 50% (Scardaci et al., 1997) and the pathogen infects rice at all developmental stages
(Ou, 1985). It is also a major problem in hybrid rice production as a result of the relatively narrow genetic base of
hybrid rice and the increased use of nitrogen fertilizer.
For disease resistance breeding in hybrid rice, conventional plant breeding techniques based on
phenotypic selection is laborious, time consuming and highly influenced by environment in comparison to Marker
Assisted Selection (MAS), which is quite precise and robust. MAS have been successfully utilized for improving
agronomic traits including disease resistance in rice (Jiang et al., 2012). In addition to improvement of pure line
varieties, MAS has also been used for improvement of parental lines of rice hybrids for resistance to blast diseases
(Fu et al., 2012). Prabhu et al., (2002) observed that the hybrids in the blast nursery were variable depending on
the degree of susceptibility resistance in the parents. When one of the parents was resistant, hybrids in general
showed complete vertical resistance even under high disease pressure.
Several blast resistance genes (Pi) demonstrated their ability in conferring resistance to different blast
pathotypes. Among them, the genes Pi1, Pi2, Pi5, Pi7, Pi9, Pi33, Pib, Pik, Pik-p, Pita, Pita2, Pizt, Pikh and Pi54i

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340

T. Shalini, P. Govintharaj, M. Ameenal, S. Manonmani & S. Robin

show broad spectrum of action (Tacconi et al., 2010). Piz was originally identified in the cultivar Zenith (Kiyosawa, 1967)
and is a broad spectrum Pi gene that has been effectively used by rice researchers throughout the world, generally found
in temperate japonica varieties.
CB 174 R is the restorer line of TNAU CMS 23 A. CORH 4 is a promising medium duration quality hybrid
derivative of the cross TNAU CMS 23 A CB 174 R maturing in 130-135 days. This hybrid has tall stature, high tillering
habit, long droopy panicles and medium slender grain type. It recorded a mean productivity of 7348 Kg/ha in five years of
trials with 17.2 per cent and 13.6 per cent increase over CO (R) 49 and the private hybrid 27 P11. At Chitlapakkam in
Kancheepuram district, this hybrid culture has recorded the highest yield of 11250 kg/ha among the trials conducted
demonstrating the highest yield potential of this hybrid. This hybrid was notified for eight states in India. The study was
undertaken to introgress the blast resistant gene into CB174 R, restorer line through Marker Assisted Breeding.

MATERIALS AND METHODS


The experiment was conducted at Paddy Breeding Station, Tamil Nadu Agricultural University, Coimbatore,
during the year of 2011-2013.
Plant Materials and Breeding Approach
The donor parent Zenith (The Piz gene derived from the USA indica rice variety) for the blast resistance was used
as a source to intogress into CB174 R the restorer line of released rice TNAU rice hybrid CO 4 (COMS 23A CB 174 R),
a medium slender grain type which matures in 135 days. The seed materials were obtained from Paddy Breeding Station,
Tamil Nadu Agricultural University, Coimbatore. Zenith showing resistance to blast with score 3 (The Piz gene derived
from the USA indica rice variety) was used for hybridization with restorer line CB 174 R. The F1 plants of CB 174R x
Zenith were genotyped for the presence of Piz. Finally the selected plants are tagged and allowed for selfing. The F2 of CB
174 R Zenith cross was subjected to both genotyping and phenotyping. In F2 population of CB 174 R Zenith, 36 plants
were chosen for the artificial inoculation of disease cultures and evaluation for resistance was done 2 weeks after
inoculation by visual score. The resistant plants were selfed to generate F3 generation. A total of one donor parent, the
recurrent parent and eight resistant lines of cross CB 174R / Zenith (F3 seeds collected from each F2 plants) were screened
along with susceptible check CO 39 under natural condition at the Hybrid Rice Evaluation Centre, located at Gudalur in
Ooty, Tamil Nadu, India during Kharif 2013 (Figure 1,). This centre is situated at an elevation of 1500 m MSL and between
11.5 N latitude and 76.5 E longitudes. The average rainfall is more than 2000 mm per year. The micro-climate at the site
favors local rice blast disease development.

METHODS
Screening Under Artificial Inoculated Conditions
The artificial blast screening was performed for parents and their hybrids (F1) and, F2 individuals along with
susceptible check by Union Sand witch method (DRR, 2011) in Department of Rice, Tamil Nadu Agricultural University,
Coimbatore. Blast lesions were isolated from diseased individuals and surface sterilized with 0.1% HgCl2 for 1 min and
placed over glass slides kept in sterile Petri-dishes padded with moist cotton and incubated for 48 h at room temperature
(28 20c). Conidia were identified from the sporulating lesions using stereomicroscope and mass multiplied in the host of
maize stem bits, cut into small pieces were placed in 50 ml Erlenmyer flasks and sterilized at a pressure of 1.4 kg /cm2 for
1 h and 30 min. Each flask was inoculated with two 5-mm dia mycelia discs of the isolate and incubated for 15 days at
Impact Factor (JCC): 4.8136

NAAS Rating: 3.53

Improving Blast Resistance in Parental Line of Rice


Hybrid through Marker Assisted Selection

341

room temperature. The stem piece was placed in a 2 lit clean & sterilized glass vessel containing 1lit of sterile water,
shaken well to dislodge the spores and decanted. The spores were used to spray on the Blast nursery using a small Ganesh
sprayer.
Observations Recorded
The test entries were scored based on leaf blast severity following SES scale (2011-2012) given here under. Two
readings were recorded on entries at 10 days intervals from 25 to 30 DAS and the maximum grade recorded in the
individual plants of each entry was taken as score for that accession
Natural Screening at Gudular
Raised beds were prepared (10 m long & 70 cm width & 10 cm height) and the top of beds were leveled
uniformly with clay paste, ensuring that there were no undulations on the beds. Then neat furrows (depth 2cm) were
formed parallel to the width of the beds (length of furrows 70 cm) at 10 cm gap, similarly, all the four sides furrows were
also formed. On the furrows the F3 seeds collected from each resistant F2 plant were sown and covered with dry red earth
so that, the seeds will not be dislodged from the furrows. To adequately induce blast disease infection, a highly susceptible
variety, CO39 was planted at both sides of F3 population (CB174R Zenith) in two rows. Plants in each row were scored
at seedling stage, tillering stage and heading stage respectively (about every 35 days) based on their lesion length.
The scoring on 1-9 scale is done as per Standard Evaluation System of the International Rice Research Institute,
Philippines
Table 1: SES Scale for Leaf Blast
Score
0
1
2
3
4
5
6
7
8
9

Description
No lesions
Small brown specks of pinhead size without sporulating centre.
Small roundish to slightly elongated, necrotic grey spots, about 1-2 mm in diameter with a distinct
brown margin and lesions are mostly found on the lower leaves
Lesion type is the same as in scale 2, but significant number of lesions are on the upper leaves
Typical sporulating blast lesions,3mm or longer, infecting less than 2% of the leaf area
Typical blast lesions infecting 2-10% of the leaf area
Blast lesions infecting 11-25% of the leaf area
Blast lesions infecting 26-50% of the leaf area
Blast lesions infecting 51-75% of the leaf area
More than 75% leaf area affected

DNA Isolation and PCR Amplification


DNA from 45 day old plants was extracted using CTAB method. Fore ground selection was carried out using
molecular marker. A molecular marker linked to blast resistance gene RM549 is used. PCR amplifications was performed
in 200 L micro centrifuge tubes containing 15 L of reaction mixture consisting of 5 ng (check) of template DNA, 0.2 M
each of forward and reverse primer, 0.25 mM each dNTPs (ATP, GTP, CTP and TTP), 0.3 mM of MgCl2, 0.75 L of Taq
polymerase (Bangalore Genei Pvt. Ltd. Bangalore) and 1x reaction buffer (10mM TrisHCl, pH 9.0, 15 mMKCl (pH 8.3)
and 1.5 mM MgCl2).
The PCR amplification was carried out in Mycycler (Bio Rad Laboratories, California) programmed for three
primers, an initial denaturation at 94C for 5 min, 35 cycles of denaturation at 94C for 1 min, annealing at 55C for 1 min,
extension at 72C for 1 min and followed by final extension at 72C for 7 min and then held at 4C. 10 L each of the
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T. Shalini, P. Govintharaj, M. Ameenal, S. Manonmani & S. Robin

product were loaded and subjected to electrophoresis in 1x TBE buffer (0.9 M Tris-HCl, 0.025 M EDTA Na2+, 0.9 M Boric
acid) at 90V. A 100 bp DNA ladder was spotted on each gel as fragment length. The gels were documented using Alpha
Imager TM 1200 Documentation and analysis system (Alpha Innotech Corporation, USA).
The individuals showing the banding pattern similar to the parent Zenith were scored as ++, the heterozygous
were scored as +-, and the plants with the alleles similar to the parent, CB 174 R were scored as --.

RESULTS
With the help of one SSR marker (RM 549), seven plants (having the Piz resistance gene in heterozygous
condition) were selected in F1 generation (Plate 1) and allowed for selfing.
In F2 population of CB 174 R Zenith, 36 plants were chosen for the artificial inoculation of disease cultures and
evaluation for resistance was done 2 weeks after inoculation by visual score (Plate 2). The disease resistance was scored
from 0 (no lesions) to 9 (necrosis of all leaves and sheaths) using SES scale method from Directorate of Rice Research
(DRR). Plants with scores of 0-3 were assigned to the resistant group, which showed elongated, necrotic grey spots, about
1-2 mm in diameter with a distinct brown margin and lesions are mostly found on the lower leaves and upper leaves. Plants
with the scores of 4-6 were assigned to medium resistant, here the blast lesions infecting 11-25% of the leaf area. Plants
with the scores of 7- 9 considered as susceptible whereas the leaf area was affected by 51-75 % of blast lesions. More than
9 was considered as a highly susceptible group of resistance, it shows 75% affected leaf area. 36 plants
(CB 174 R Zenith) were subjected to PCR analysis using DNA marker RM549 in F2 generation. These markers linked to
resistance genes allowed efficient screening of the F2 generation. Scoring was done based on the banding pattern with
reference to their parents, that is those bands similar to resistant and susceptible parents were scored as + and respectively, while the plants having bands from both parents were scored as +-. Based on genotypic data and phenotypic
data 18 plants were showed moderately resistance, 8 plants were assessed resistance and 13 plants were showed susceptible
(Table 1). The homozygotes and heterozygotes scored and goodness of fit was tested using chi square value for the
segregating data. In (CB 174 R Zenith), the chi square test showed non significance at the (5% level) in Piz, out of 36
plants 23 plants were showed the resistant and 13 plants were showed susceptible (Table 2). Segregation pattern of resistant
and susceptible plants fits well with 3:1 ratio. Identified positive plants (resistant) were screened for Rf genes. Among
these, resistance plants with Rf gene were selected and selfed for evaluation during next season. In F2 generation eight
plants showed resistance to blast is allowed for selfing to generate F3 generation. F3 seeds of eight resistant plants were
subjected to natural screening at Gudalur (Plate 3). Among eight lines 2 lines were found to be resistant, 2 lines were found
to be moderately resistant and 4 lines were found to be susceptible (Table 3).

DISCUSSIONS
A total of 36 plants (CB 174 R Zenith) were subjected to PCR analysis using DNA markers. These markers
linked to resistance genes allowed efficient screening of the F2 generation. Scoring was done based on the banding pattern
with reference to their parents. Based on genotypic data and phenotypic data, 18 plants showed moderately resistance, 8
plants were assessed resistance and 13 plants were showed susceptible. The observed and expected frequencies of the
various marker genotypes were calculated using the chi square test. The homozygotes and heterozygotes scored and
goodness of fit was tested using chi square value for the segregating data. In CB 174 R Zenith, the chi square test showed
non significance at the 5% level.
Impact Factor (JCC): 4.8136

NAAS Rating: 3.53

Improving Blast Resistance in Parental Line of Rice


Hybrid through Marker Assisted Selection

343

Out of 36 plants, 23 plants were showed the resistant and 13 plants were showed susceptible. Segregation pattern
of resistant and susceptible plants fits well with 3:1 ratio confirming the fact of resistance controlled by single gene.
Padmavathi et al., (2005) also reported the same ratio for Piz gene for the cross CO39 Zenith.

CONCLUSIONS
Breeding lines are evaluated in two ways: in greenhouse inoculations and in outdoor nurseries. Greenhouse tests
are valuable for characterizing pathotype-specific resistance, which usually is of a hypersensitive type. Because of their
monocyclic nature and highly variable temperature-light-moisture conditions, greenhouse tests are not reliable indicators of
partial resistance. Outdoor nurseries provide opportunities to observe disease development over time with polycyclic
exposure of test lines to infection. These tests give a relative measure of field or partial resistance to leaf blast, assuming a
compatible host-pathogen combination. The HREC field in Gudalur prefecture possess suitable for field conditions for
blast disease development and were used in July 2013 for evaluation of leaf blast resistance. Among 8 lines of CB 174R/
Zenith, 2 lines were resistant to leaf blast. Ishihara et al., (2014) developed an F2 population from a cross between
Miyazakimochi and Bikei 22 and F3 lines were used to characterize the panicle and leaf blast resistance of Miyazakimochi
in Dasein in year 2000. F3 lines which are resistant to blast under natural condition are to be evaluated for different isolates
of blast pathogen to confirm major gene (Piz) resistance. This material will be evaluated for further generation along with
restorability and will be used for developing high yielding rice hybrids with blast resistance.
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Ishihara, T., Y. H. Saito, S. Oide, K. Ebana, N.Tuan La, K. Hayash, T. Ashizawa, F. Suzuki and S. Koizumi. 2014. Quantitative
trait locus analysis of resistance to panicle blast in the rice cultivar Miyazakimochi. Rice.7:2

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Jiang, H., Y. Feng, B. Liang, X. Li, G. Gao, Q. Zhang, X. Jinghua, C. Xu and Y. He. 2012. Improving blast resistance of Jin 23B
and its hybrid rice by marker-assisted gene pyramiding. Mol Breeding., 30:16791688.

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Joseph, M., S. Gopalakrishnan, R.K. Sharma, A.K. Singh, V.P. Singh, N.K. Singh and
bacterial blight resistance and Basmati quality

T.Mohapatra.

2004.Combining

characteristics by phenotypic and molecular marker assisted selection in

rice. Mol. Breed., 13:377387.


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Kiyosawa, S. 1967. Genetic studies on host-pathogen relationship in the rice blast disease. Proc.

Symp. Rice diseases and

their control by growing resistant varieties and other measures,Tokyo. pp. 137153.
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Ou,S.H. 1985. Rice diseases. 2nd ed. Common w.Mycol. Inst., Kew, Survey, England, 38

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Padmavathi, G., T. Ram, K. Satyanarayana and B. Mishra. 2005. Identification of blast (Magnoporthe grisea) resistance genes
in rice. Curr Sci., 88:628630

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Prabhu, A.S., E.P.Guimres, M.C. Filippi, L.G. Araujo & V.Cutrim. 2002. Expression of resistance in rice hybrids to
Pyricularia grisea. Fitopatologia Brasileira. 27:454-460.

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APPENDICES

Figure 1: Introgression of BB Resistance into CB 174R

Plate 1: Identification of Heterozygotes in F1 (CB 174R/Zenith)

Impact Factor (JCC): 4.8136

NAAS Rating: 3.53

Improving Blast Resistance in Parental Line of Rice


Hybrid through Marker Assisted Selection

345

Plate 1: Artificial Phenotypic Screening for Blast

Plate 3: Evaluation of Blast Screening Nursery at HREC, Gudalur


Table 2: Screening of Blast Resistance in F2 s of CB 174 R Zenith under Artificial
Inoculated Condition (Sandwitch and Spore Suspension Spraying)
Genotype
1
2
3
4
5
6
7
8
9
10
11
12
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Piz
++++
-+++
++
++-++-

Scale
4
5
3
7
5
3
2
5
5
7
5
4

Reaction to genotype
Moderately resistance
Moderately resistance
Resistant
Susceptible
Moderately resistance
Resistant
Resistant
Moderately resistance
Moderately resistance
Susceptible
Moderately resistance
Moderately resistance
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T. Shalini, P. Govintharaj, M. Ameenal, S. Manonmani & S. Robin

13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Zenith
CB174 R
CO 39
( Susceptible check)

Table 2: Contd.,
-7
-7
+5
-7
-7
-7
+3
+5
++
2
++
3
-7
-7
+5
+5
++
2
-7
-7
-7
+5
-7
++
2
+5
-7
-7
3
7
9

Susceptible
Susceptible
Moderately resistance
Susceptible
Susceptible
Susceptible
Resistant
Moderately resistance
Resistant
Resistant
Susceptible
Susceptible
Moderately resistance
Moderately resistance
Resistant
Susceptible
Susceptible
Susceptible
Moderately resistance
Susceptible
Resistant
Moderately resistance
Susceptible
Susceptible
Resistant
Susceptible
Susceptible

Table 3: Chi-Square Test for F2 Population for the Cross of CB 174 R Zenith for Piz Gene
Gene

Piz

Marker

RM 549

Reaction

Genotypic Data
Observed Expected
Value (O) Value(E)

Blast
Resistant
Marker
Blast
Susceptible
Marker

23

(O-E)

(O-E )2

(O-E )2 /
E

-4

16

0.59

27

2
Value
2.37N
S

13

16

1.78

Phenotypic Data
Piz

RM 549

Resistant
plants
Susceptible
plants

21

27

-6

36

1.3

15

36

4.0

5.3 S

Table 4: Reaction of Blast Resistance in F3s of CB 174 R Zenith under Natural Condition
S.No
1
2
3

Impact Factor (JCC): 4.8136

Score
3
5
7

Reaction
Resistant
Moderately Resistant
Susceptible
Total

No. of Lines
2
2
4
8

NAAS Rating: 3.53

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