Breeding for bph resistance in rice.

1. SEMINAR ON
BREEDING FOR BPH RESISTANCE
IN RICE
Presented by :
Ankita Jena
Admn. No. : 08PBG/16
MSc.(Ag) 2nd year
Dept. of Plant Breeding And Genetics

2. INTRODUCTION
RICE
• Rice is the major staple crop for about half of the worlds
population. It is the primary source of calories for more
than one–third of the world population.
• It is predicted that an additional 116 million tons of rice
will be needed by 2035 to feed the growing population
(Seck et al. 2012).
• However, the brown planthopper (BPH, Nilaparvata
lugens Stål), which sucks the phloem sap of the rice leaf
sheath and transmits viral diseases such as rice grassy
stunt virus (RGSV), rice ragged stunt virus (RRSV) and
rice wilted stunt virus (RWSV), often leads to severe yield
losses in the agricultural industry (Fujita et al. 2013).

3. • India rank second in production of rice after China.
• Up to 60% yield loss is common in susceptible rice
cultivars attacks by Brown plant hopper (BPH)
• The main method of controlling BPH is application of
pesticides such as imidacloprid but indiscriminate use
of chemicals leads to environmental pollution, kills
natural enemies of the target pest and may result in
development of resistant/tolerant races of BPH.
(Lakshmi et al. 2010; Tanaka et al. 2000).
• Host-plant resistance is therefore most desirable and
economic strategy for the control or management of
BPH (Jena et al. 2006).

4. Some Major Economic losses due to
BPH
 1732 Japan famine – death of about 1 million people
 Due to severe attack of BPH on rice crop (Kaneda 1978)
 1973-74, Kerala state of India
 50,000 ha of rice were severely damaged
 8,000 ha of rice crop totally wiped out by the insect.
 2005, China reported a loss of 2.7 mt of rice due to direct
damage by BPH
 0.5 mt of rice in Vietnam damaged due to indirect losses by
viruses transmitted through BPH (Brar et al. 2010)
2017: In Odisha, India
19,904 hectares of land were destroyed due to the insect attack
among which 13,009 hectares (65%) area were destroyed by brown
plant hopper (BPH) alone.

5. INTRODUCTION contd….
BROWN PLANT HOPPER
( Nilaparvata lugens)
▫ Kingdom: Animalia
▫ Phyllum: Arthropoda
▫ Class: Insecta
▫ Family: Delphacidae
▫ Order: Homoptera
▫ Genus: Nilaparvata
▫ Species: N. lugens
• One of the serious pests of rice crop
in Asia pacific region causing about
10-70% yield loss.
• The BPH is believed to have undergone a
host shift from Leersia (cut grass) plants to
rice about 0.25 million years ago. After that,
BPH evolved as a monophagous insect
herbivore of the cultivated rice.
( P.L Jones et al 1996)
Hopper burn

6. Nature of BPH damage
One of the most destructive insect pests causing significant
yield loss (10-70 %) in most of the rice cultivars (Sogawa et
al. 2003)
Damages the plant
 by sucking the phloem sap
 causes hopper burn
 transmits viral diseases
Grassy stunt virus (RGSV)and
Ragged stunt virus(RRSV)
 Rice wilted stun virus

7. HOW TO AVOID SUCH
HUGE LOSS ?

8. Host-plant resistance
Identification of resistance genes is imperative
Cultivated
gene pool
Wild
relatives
To tackle drawbacks of conventional
measures and the evolution of new
biotypes
6

9. RELATION BETWEEN BIOTYPES OF
BPH AND RESISTANCE GENES
The biotypes of BPH show clear differences in
virulence pattern on rice cultivars/genotypes. Four BPH
biotypes are known for rice.
Biotypes 1 and 2 : widely distributed in South east and
East Asia
Biotype 3 was developed in the laboratory by rearing
the insects on the resistant variety ASD7 which has the
bph2 gene for resistance (Panda and Heinrich 1983).
Biotype 4 (most destructive) : Occurs in the Indian
subcontinent and it is also called South Asian biotype.

10. VARIETY/SOURCE GENE REACTION TO BIOTYPES
1 2 3 4
Mudgo Bph1 R S R S
ASD 7 Bph2 R R S S
Rathu Heenati Bph3 R R R R
Babawee Bph4 R R R R
ARC10550 Bph5 S S S R
Swarnalata Bph6 S S S R
T12 Bph7 S S S R
Chin Saba Bph8 R R R -
Balamawee Bph9 R R R -
TN1 none S S S S
O.Officinalis Bph6, bph13 D R R R R
O.Minuta Bph20 , bph21 R ND ND ND
O.latifolia Bph12 ND R ND ND
O.australiensis Bph18 R R R R

11. Evaluation of BPH resistance
• Based on the type host:pest interaction, evaluation methods
can be divided into two groups.
• The first evaluates host resistance by measuring the degree of
damage following BPH infestation.
▫ Seed box test : It is recognized as a standard method which
assesses damage to seedlings caused by the progeny of an initial
infestation with a set number of nymphs (Panda and Khush
1995).
▫ Advantage : time- and space-saving assay .
▫ Disadvantage :affected by temperature, humidity, nymphs instar,
density, biotype, population and natural enemies.
• The second approach indirectly the relative host response by
examining the physiological and biochemical reactions of the
BPH (feeding rate, fecundity and survival) feeding on
different varieties.
▫ Parameters measured : Honeydew excretion, survival rates,
preference settling, and feeding behavior.

12. Source of BPH resistance
• The Genesy database
maintained at IRRI has 573
cultivated rice accessions that
showed resistance to at least
one BPH biotype.
• Among these 484 accessions
(92.5 %) showed resistance to
biotype 1.
• Only 80 accessions (15.3 %)
were resistant to all three
biotypes (Fig. 1).
• Eighteen species of wild rice,
comprising 265 accessions,
were highly resistant, and two
species (O. officinalis and O.
minuta) accounted for 41 % of
the total (Fig. 2). Fig. 1

13. Fig. 2

15. GENETICS OF BPH
RESISTANCE

16. Mapped BPH resistance genes
• Thirty one BPH resistance genes have been identified
from ssp. indica and wild relatives.
• Twenty nine BPH-resistance genes have been located on
the chromosomes of rice.
• Most of these genes were located to specific rice
chromosome regions, but the identities of a few (e.g.
bph5 and bph8) are confusing.
• To date more than ten genes have been fine mapped to
regions of less than 200 kb.
• Most of resistance alleles are dominant, but few are
recessive (bph4, bph5, bph7, bph8, bph19 and bph29).

19. Mapping of minor BPH resistance QTLs
• Using different mapping populations (RIL, DH, F2:3) more QTLs were
detected on all rice chromosomes except 5 and 9 .
• Studies showed that some highly resistant varieties carry many minor
QTLs along with one or more major genes (gives durable resistance).
▫ Example : IR64 from IRRI showed more durable resistance than IR26,
although both carry Bph1 because of seven minor QTLs were detected
on chromosomes 1, 2, 3, 4, 6 and 8 of IR64 (Alam and Cohen 1998).
▫ Sri Lankan variety Rathu Henati has shown durable resistance to all
four BPH biotypes in Southeast Asia since the 1970s, as it not only
carries major genes Bph3 and Bph17, but also minor QTLs on
chromosomes 2, 3, 4, 6 and 10 (Jairin et al. 2007; Kumari et al. 2010;
Sun et al. 2005).
▫ Similarly indica cultivar ADR52 possesses two major genes Bph25 and
Bph26, along with several minor QTLs associated with resistance to
BPH, white-backed planthopper (WBPH) and green leafhopper
(Srinivasan et al. 2015).

20. Map based cloning of BPH resistance
genes
• To date, Bph14, Bph26, Bph17 and bph29 have been
cloned by map-based cloning.
• Bph14 is the first cloned BPH resistance gene originated
from O. officinalis. Bph14 was originally fine-mapped to
a 34 kb region on chromosome 3 L.
• Bph14 encodes a coiled-coil, nucleotide-binding and
leucine-rich repeat (CC-NB-LRR) protein which might
function in specific recognition of a BPH effector, with
activation of the defense response through induction of a
SA-dependent resistance pathway (Du et al. 2009).

22. Bph 26
Cloned from ADR52( carries two genes, Bph25 and Bph26) located on
chromosomes 6S and 12 L, respectively (Myint et al. 2012).
Bph17
Cloned from Sri Lankan indica variety Rathu Heenati. Bph17 is actually a
cluster of three genes encoding plasma membrane-localized lectin receptor
kinases (OsLecRK1—OsLecRK3), which collectively function to confer
broad-spectrum, durable resistance and provide an important gene source for
MAS and transgenic breeding for BPH resistance (Liu et al. 2015).
Bph29
Cloned from O. rufipogon, fine-map to a 24 Kb region on chromosome 6S.
Bph29 encodes a B3 DNA-binding protein. It is restricted to the vascular
tissue where BPH attacks. Expression of bph29 activates the SA signaling
pathway and suppresses the jasmonic acid/ethylene (JA/Et)-dependent
pathway after BPH infestation and induces callose deposition in phloem
cells, resulting in antibiosis to BPH (Wang et al. 2015).

24. Genes and TFs associated with BPH
resistance
• Some genes and transcription factors (TFs) associated
with BPH resistance have been identified through reverse
genetics approaches such as T-DNA mutants and genes
homology.
• Bphi008a is a resistance gene that is induced by BPH
feeding; it is involved in ethylene signaling. Plants
carrying a transgenic Bphi008a allele show significantly
enhanced resistance to BPH (Hu et al. 2011).
• Another two genes, OsERF3 and OsHI-LOX, are
ethylene response factors and lipoxygenase genes,
respectively, involved in a JA/Et-dependent pathway and
act as inhibitor of the gene expression to improve
resistance to BPH (Lu et al. 2011; Zhou et al. 2009).

26. Basis of BPHresistance
Cloned genes
Bph 14
(O.officinalis)
Bph 26
(ADR 52)
Genetic transformation
Mediate deposition of callose in phloem cells causing
sucking inhibition
Transgenic rice expressing Bph 14 gene causes elevated expression of
salicylic acid synthesizing genes (just like in plant-pathogen interactions)
Tamuraetal. 2014;Wangetal.2015

27. Volatile chemicals mediating BPH
resistance
Linalool (attracts BPH
predators)
Benzyl benzoate (released
on plant surface)
Resistant varieties
produce it inabundance,
when infested byBPH
Ovicidal property
Chengetal. 2007;Sienoetal.
1996
Xiao et al., 2012

28. MABC for BPH resistance
It takes a minimum of 6–8 backcrosses to fully recover a recurrent parent
genome using conventional breeding methods, but MABC enables the
procedure to be shortened to 3 or 4 backcrosses (Tanksley et al. 1989). There
are three levels of selection in which markers are applied in backcross
breeding .
Firstly, markers are used to select target alleles whose effects are difficult
to observe phenotype (e.g. resistance in the absence of actual disease/pest
tests), this is referred to as ‘positive selection’.
Secondly, markers are used to select for progeny with the target gene and
tightly-linked flanking markers in order to produce chromosomes that harbor
the target allele with minimal surrounding DNA from the donor parent
(minimizing linkage drag), designated as ‘negative selection’ or
‘recombinant selection’.
Thirdly, markers that are distributed across all 12 rice chromosomes can be
selected for recovery of the recurrent parent genome, known as ‘background
selection’.

30. CASE
STUDY

32. EXPERIMENTAL DETAILS
Abstract : A first set of 25 NILs carrying ten BPH
resistance genes and their pyramids was developed
in the background of indica variety IR24 for insect
resistance breeding in rice.
Material methods:
Eight independent backcross breeding methods were
used with the susceptible indica rice cultivar IR24 as
the recurrent parent and eight BPH-resistant (R)
donor cultivars.

33. DONORS:
 PTB33 (BPH2 and BPH32)
 Rathu Heenati (BPH17 and BPH32)
 Babawee (BPH4)
 Pokkali (BPH9)
 IR65482-4-136-2-2 (BPH10)
 IR65482-7-216-1-2 (BPH18)
 ADR52 (BPH26)
 IR71033-121-15 (BPH20 and BPH21).
• To develop a set of nine NILs with a single R gene for BPH
resistance.
• Development of NILs
• Genotyping and validation test of NILs
• BPH bioassays of NILs for resistance or susceptibility.
• Evaluation of NILs for antibiosis resistance by honeydew
experiment.
• Evaluation for agronomic traits.
• Background genotyping

35. Result and conclusion:
• The percent recovery of the recurrent parentage genome of the 25 NILs with single
and multiple R genes ranges from 82.0 to 94.2%. However, some of the NILs
(BC2F5–BC3F5) revealed a low recovery (82.0–89.4%) of the IR24 genome,
significantly below the theoretical value of 87.5–93.75% possible in following the
two to three conventional back Crossings.
• The comparison of the agronomic performance of the NILs and the recurrent parent
IR24 based on field evaluation proved that the BPH R genes had no yield penalty.
• However, some NILs/PYLs showed a significant increase in plant height and
panicle length at the P= 0.05 level. These small differences might be caused by the
influence of the donor alleles inherited from the donor genome(s).
• Overall the agronomic performance of the NILs showed either superior
performance or performance similar to IR24 demonstrating the efficiency of the
MAB program in recovery of the recurrent parent, IR24 genome and at the same
time having the target BPH R gene(s) for resistance.
• Furthermore, the NILs with multiple R genes having two to three genes developed
in this study are quite promising materials and these lines might serve as new
genetic resources in rice breeding aimed at increasing the durability of host-plant
resistance against BPH in different rice-growing countries.

37. Abstract
• Out of 1989 wild accessions sown in seed boxes for screening,
only 1003 wild accessions with good germination were
screened against brown planthopper (BPH) under greenhouse
conditions.
• The collection comprised of accessions from 11 wild species
and African cultivated rice.
• As many as 159 accessions were identified as resistant during
the year 2012 based on one year screening. A selected set of
BPH resistant accessions were screened again during 2013.
Based on the two years screening,
▫ seven accessions of O. nivara (AA),
▫ one accession of O. officinalis (CC),
▫ seven accessions of O. australiensis (EE),
▫ five accessions of O. punctata (BB and BBCC) and
▫ nine accessions of O. latifolia (CCDD) were confirmed to be
resistant to BPH.

38. Material and Methods
Rearing of BPH colony:
• BPH biotype 4 was maintained on 30-day-old TN1 rice plants
under greenhouse conditions at 28 °C ± 2 °C, 75% ± 5%
relative humidity and 14 h light and 10 h dark photoperiod
conditions positioned at Ludhiana, India (30o54′ N and 75o48′
E) in India according to Heinrichs et al (1985).
• For obtaining the 2nd to 3rd instar nymphs, newly emerged
male and female adults were paired and released on 30-day-
old TN1 plants for oviposition.

39. Raising test genotypes
• The pre-germinated seeds of the test accessions
were sown in seed boxes (45 cm × 35 cm × 10 cm)
containing well puddled soil in rows at spacing of
3.5 cm apart.
• The sprouted seeds of susceptible check TN1 were
sown in two border rows and in half of the middle
row.
• The accessions were sown with the maximum of 10
seeds in each line depending upon availability of
seed.
• All the test plant trays were raised in an insect-
proof greenhouse at 30 °C ± 2 °C, 80% ± 5% relative
humidity.

40. Screening of genotypes:
• There were three replications for each accession and these were infested at
10–12 d old with the 2nd to 3rd instar hopper nymphs @ 6–8 nymphs per
seedling.
• Damage score on each line was recorded on 0–9 scale when more than
90% plants of TN1 were killed following the Standard Evaluation System
(SES) of rice (IRRI, 1996).
• Each accession was scored on the individual plant basis as
▫ 0 (no visible damage),
▫ 1 (partial yellowing of first leaf),
▫ 3 (first and second leaves of most of plants partially yellow),
▫ 5 (pronounced yellowing and stunting or about half of the plants wilted or
dead),
▫ 7 (more than half plants wilted or dead and remaining plants severely
stunted),
▫ 9 (all plants wilted and dead) (IRRI, 1996).
• The interpretation of results of each accession had been based on standard
evaluation system in which accessions with a mean rating of 0–3.49, 3.50–
5.49 and 5.50–9.00 were designated as resistant, moderately resistant and
susceptible, respectively (Heinrichs et al., 1985).

41. Result :
• Out of 30 designated BPH resistance genes, Bph10 and
Bph18 have been transferred from O. australiensis;
bph11, Bph13, Bph14 and Bph15 from O. officinalis;
Bph12 from O. latifolia; Bph20, Bph21 and Bph23 from
O. minuta and Bph24, Bph29 and Bph30 from O.
rufipogon.
• No BPH resistance gene had been identified in O. nivara
(AA) or O. punctata (BB). These two species thus may
represent new source for resistance to BPH.
• The seven resistant accessions of O. nivara originates
from India, Nepal and Cambodia and may have different
genes for BPH resistance.

42. CONCLUSION:
• Rice production needs to be increased by controlling the damage
caused by biotic stresses particularly BPH in terms of significant yield
loss annually.
• Host-plant resistance is an effective environment friendly approach to
reduce BPH damage and increase yield potential of cultivars.
• Although 31 BPH resistance genes have been identified, further efforts
are needed to identify new resistance genes from diverse genetic
sources which may confer resistance to new biotypes of BPH.
• Some of the known resistance genes could be pyramided and tested for
efficacy in conferring resistance to new biotypes of BPH.
• In order to achieve stable resistance to BPH, pyramided major genes
or QTLs may provide durable resistance and improve yield potential
of cultivars.
• Development of functional SNPs associated with BPH resistance could
also provide additional genomic tools to the implementation of BPH
resistance genes for improved elite rice cultivars and a stable rice
production.

43. REFERENCES
• ShengliJing & YanZhao (2013) Genomics of interaction between the brown plant hopper
and rice. Current Opinion in Insect Science 2017, 19:82–87
• Cheng, Xiaoyan et al.(2013). Towards Understanding of Molecular Interactions between
Rice and the Brown Planthopper. Molecular Plant , Volume 6 , Issue 3 , 621 - 634
• Hu J, Xiao C, He Y(2016). Recent progress on the genetics and molecular breeding of
brown planthopper resistance in rice. Rice. 2016;9:30.
• Lei Peng , Yan Zhao1, Huiying Wang1, Chengpan Song1, Xinxin Shangguan, Yinhua
Ma, Lili Zhu1 and Guangcun He. Functional Study of Cytochrome P450 Enzymes from
the Brown Planthopper (Nilaparvata lugens Stål) to Analyze Its Adaptation to BPH-
Resistant Rice, Published online 2017 Feb 28. doi: 10.1186/s12870-017-1005-7
• Changyan Li, Chao Luo,Zaihui Zhou, Rui Wang, Fei Ling, Langtao Xiao, Yongjun Lin,
and Hao Chen, 2015Jul Gene expression and plant hormone levels in two contrasting
rice genotypes responding to brown planthopper infestationJ Plant Physiol. 1;183:13-22.
doi: 10.1016/j.jplph.2015.03.020. Epub 2015 May 27.
• Du B, Wei Z, Wang Z, Wang X, Peng X, Du B, Chen R, Zhu L, He G, Y. Xiao, Q. Wang,
M. Erb, T.C.J. Turlings, L. Ge, L. Hu, J. Li, X. Han, T. Zhang, J LuSpecific herbivore-
induced volatiles defend plants and determine insect community composition in the field
Ecol. Lett., 15 (2012), pp. 1130-1139