The presentation covers overall aspects of Breeding for BPH resistance in rice. It was used latest literature for preparing this. This presentation would be great help to any interested person on BPH resistance of rice

1. Breeding for BPH resistance in rice, new
sources and approaches
K.A.K.Wijesena
Assistant Director of Agriculture(Research)
RRDI, Batalagoda, Sri Lanka
(Online seminar-2021)

2. Outline
• Introduction
• Mechanisms for BPH resistance
• Methods for BPH screening
• BPH biotypes
• Sources of BPH resistance
• Genes of BPH resistance
• Novel approaches for BPH resistance
• Conclusion and perspective
• References

3. Introduction
• Various biotic and abiotic stresses decreases rice productivity of most
rice growing countries
•Approximately 52% global rice production lost annually due to biotic
stresses
•Out of the total 25% due to insect pests (Yarasi et al.,2015)
•Among the biotic stresses, brown planthopper is most devastating
insect for rice cultivation
• It is a sap sucking insect and damage cause by adults or nymph
• severe infestations cause “hopper burn’’
• transmit virus diseases grassy stunt and ragged stunt
• yield losses- 20% -80% in sensitive cultivars

4. • BPH damage cause reductions in,
 leaf area
 photosynthetic rate
 stem nitrogen concentrations
 chlorophyll contents
 dry matter accumulation
 yield
• In the field, feeding by large numbers of BPHs usually causes
‘‘hopperburn”, It can be identified by
 drying of the leaves
 wilting of the tillers of susceptible rice varieties

5. Factors that favour BPH development
• Adoption of high yielding BPH sensitive varieties
• Higher humidity (greater than 80%)
• Temperature of about 25-30 0C
• Application of nitrogenous fertiliser beyond the recommended
rate
• Higher plant density
• Stagnating water in the field
• Ineffective application of pesticide at early stage of crop
growth which destroy natural enemies
Therefore, regular monitoring of rice crop is the only way for
effective BPH management.

6. • Unusual weather changes cause severe outbreaks (Optimum
Temperature, High RH, low wind movement)
• An average of 5-10 % of rice lands of the Sri Lanka affected
annually due to BPH damage(Nugaliyadde et al., 2001)
• Pesticide is most commonly used as control measure
• High production cost, potentially harmful to the environment,
human health and destroy natural enemies
• Host resistance in rice is economical and effective measure
• Therefore, Identification and utilization of resistance sources
and application of novel molecular approaches are most
effective strategy for BPH management

7. Scientific classification
• Kingdom – Animalia
• Phylum – Arthropoda
• Class – Insecta
• Order – Hemiptera
• Family – Delphacidae
• Genus – Nilaparvata
• Species - lugens

8. • Host plant
 BPH is monophagous- feeding only on rice
 However, in no choice condition, BPH will feed on finger millet,
sugarcane, maize, echinochloa, cyperus, sorghum and wheat
• Distribution
 BPH is the most economically important plant hopper in Asia
 It occurs throughout India, Sri Lanka, Bangladesh, China, Indonesia
and Philippines

9. Mechanisms for BPH resistance
• Antibiosis
resistance mechanism by which the host plant leads to
injury or mortality, decreased growth,longevity, reduction
of pest reproduction.
• Antixenosis or non preference
host plant exhibits features that are unattractive or desirable
for the insect pest to invade or damage. (allelo-chemicals,
volatile, colour and texture)
• Tolerance
Plant is able to recover or withstand insect pest damage
while manifesting infestation symptoms

10. Resistance mechanisms of BPH resistant donor varieties
(Haliru et al., 2020)
Variety Gene function BPH
reaction
Swarnalata Antibiosis R
T12 Antibiosis/tolerant HR
Ptb 33 Antibiosis R
Rathu heenati Antibiosis/antixenosis R
Pokkali Antibiosis/ tolerant MR
ASD 7 Antibiosis/tolerant MR
Babawee Antibiosis/antixenosis R
Mudgo Antibiosis/antixenosis R
IR 64 Antibiosis/tolerant MR
Kolayal Antibiosis R
Polayal Antixenosis/antibiosis R

11. Methods for BPH screening in rice
• Standard seed-box screening test
• Honey dew test
• Nymphal survival method
 Ptb 33- Resistant check
TN 1, Bg 380(Local)- susceptible check

12. Preparation of
seedling trays
Seed sowing /2-4 replicates
Seedling emergence
Evaluation according to IRRI
standard (one week after
infestation)
Infestation of 5 th nymphal stage of
BPH (10 day old seedlings)
Standard Seed Box
Method

13. Evaluation of BPH resistance
Symptoms Scale
(for greenhouse test)
Reaction
•Partial to pronounced
yellowing and
increasing of stunting
•Extreme signs are
wilting to death of plants
•Infested areas in the
field may be patchy
0- No injury R
1- Very slight injury R/MR
3- First and 2nd leaves of
most plants partially yellowing
MR
5- yellowing and stunting or
about 10-25% of plants
wilting or dead and remaining
plants severely stunted or
drying
MR/MS
7- More than half of the
plants wilting or dead
MS
9- All plants dead S
Source: Standard Evaluation System for Rice( IRRI), 2014

14. Honey dew test
• 30 days old seedlings with
first tiller used
• Filter paper stained by
bromocresol green(0.02%)
2-3 hoppers (starved
for 3-4 hrs) release
into the chamber
BPH allow to
feed for 24 hrs
honey dew indicated as
blue rimmed spots
• area of each spot can be
measured using 1x1mm grid/
digital scaner
•Statistical analysis
(SAS,Image-J software version
1.48)

15. Nymphal survival method
• Kept 20 newly hatched nymphs in a pot with 3 rice plants (40
days old) placed in mylar cages
• The number of surviving nymphs recorded every two days
until they became adults
• Three reps with control plants will be maintained
• Number of nymphs survived on resistant plants are lower
(Sai Harini et al, 2013)
(Harini et.al,2013)

16. BPH biotypes
• There are four predominant BPH biotypes found across the
major rice growing countries
• Biotype 1& 2 – widely distributed in south east and east asia
• Biotype 3 – developed by rearing the insects on the resistant
variety (ASD 7) which has the bph 2 gene in the laboratory at
IRRI and Japan
• Biotype 4 (most destructive) – occurs in the Indian
subcontinent and also called as south asian biotype
 No comprehensive studies carried out in SL to identify BPH
biotypes
 Fernando et.al,2007 reported, BPH population found in
Kegalle area is genetic variant from the Batalagoda population
using molecular analysis

17. Sources of BPH resistance
Cultivated rice
At IRRI, by mass screening
identified (Jackson,1997)
• 573 cultivated rice
accessions-resistance to
at least one biotype
• 484 acc (92.5%) showed
resistance to biotype 1
• only 80 acc (15.3%)
resistance to all 3
biotypes.
(https://www.genesys-
pgr.org)

18. Relationship between biotypes of brown planthopper and
resistance genes from diverse sources
Variety/Source Gene Reaction to biotypes
1 2 3 4
Mudgo Bph1 R S R S
ASD7 bph2 R R S S
Rathu heenati Bph3 R R R R
Baba wee bph4 R R R 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.minuta (acc. 101141) Bph20,Bph21 R ND ND ND
O.latifolia (B14) Bph12 ND R ND ND
O. officinalis (acc.100896) Bph6,Bph13 R R R R
O. australiensis (acc. 100882) Bph18 R R R R
R-Resistant S- Susceptible ND- No data
(Jena et al., 2010)

19. Sources from wild rice
• Eighteen spp. of wild rice, comprising 265 accessions, found to be highly resistant
• two species (O. officinalis and O. minuta) accounted for 41% out of the total
(Jie Hu et.al,2016)

20. Genes of BPH resistance
 To date, 38 BPH resistance genes have been identified from
spp. Indica and wild relatives.(Kumar et.al,2020)
 Most of these genes were located to specific rice chromosome
regions
 To date more than 10 genes have fine mapped to regions of
less than 200kb
 Most of resistant alleles are dominant
 Few are recessive (bph4,bph5,bph7,bph8,bph19 and bph29)

21. Resistance of Asian cultivars carrying BPH resistance genes
( Jie Hu et al, 2020)
Name Acc no.
(IRRI)
Origin Gene R S (seedling
stage)
RL
MGL2 6218 IND Bph1 3.0 R
IR 28 30411 PHL BPh1 4.0 MR
IR 29 30412 PHL Bph1 7.47 MS
IR 30 30413 PHL Bph1 7.14 MS
IR34 30415 PHL Bph1 4.12 MR
IR26 24154 PHL Bph1 2.83 R
IR 44 39341 PHL Bph1 2.56 R
IR 36 39292 PHL bph2 2.5 R
PTB 18 11052 IND bph2 1.8 HR
Gangala 15259 SL Bph3 3.01 MR
Mudukiriel 15719 SL Bph3 5.46 MS
Hondarawala 31415 SL Bph3 1.86 HR
Muthumanikkam 40850 SL Bph3 1.46 HR
Kuru Hodarawalu 36303 SL Bph3 3.00 R
Babawee 8978 SL bph4 1.50 HR
Lekam samba 15412 SL bph4 2.92 R
Sulai 15421 SL bph4 3.25 MR

22. major BPH resistance genes identified from wild species
Resistance Gene Chromosome source
Bph 10 12 L O. australiensis
Bph 11(t) 3L O. officinalis
Bph 12 4 S o. latifolia
Bph 13(t) 2L O. eichingeri
Bph 13(t) 3S O. officinalis
Bph 14 3 L O. officinalis
Bph 15 4S O. officinalis
Bph 18(t) 12 L O. australiensis
Bph 18(t) 4L O.rufipogon
Bph 20, Bph 21 4S, 12L O. minuta
Bph 34 4L O.nivara
Bph 36 4S O. rufipogon
(Kumar et al,2020)

23. • The donor varieties which having Bph1,bph2,Bph3 and Bph4
genes were extensively used to develop IR varieties
• About 60% of the SL varieties and 10% Indian cultivars
posses the bph 2 gene (Jena et al,2010)
• Ptb 33 principal donor parent in our breeding program

24. Map based cloning of BPH resistance genes
• To date, 8 genes (Bph14,Bph26,Bph17,Bph29,Bph18,Bph32,Bph9
and Bph6) have been cloned by map based cloning.
• Bph 14 is the first cloned Bph resistant gene originated from O.
officinalis. It was fine-mapped to a 34kb region of chr 3L
• Bph 26 cloned from indica variety ADR 52 (carries Bph 25 and Bph
26) located on chr 6S and 12L
• Bph 17 cloned from SL indica variety Rathu Heenati and fine mapped
to 79kb region on chr 4S
• Bph 29 recessive gene from O. rufipogon, fine mapped to 24kb region
on chr 6S.

25. Five BPH resistant gene clusters
(Haliru et al,2020)

26. Location of map-based cloned BPH resistance
genes on chromosomes
(Jie Hu.et al., 2016)

27. Marker Assisted Selection
• conventional breeding-takes minimum of 6-8 backcrosses to
fully recover a recurrent parent genome
• MABC shorten the breeding cycle for 3-4 backcrosses
• It reduces the genome content of the pollen parent into the
genetic makeup of recipient parent.
• MAS has widely applied for identification and introgression of
BPH resistance genes into susceptible cultivars
• Using MAS can combine multiple genes/QTLs together into a
single genotype simultaneously
• The fundamental prerequisite of MAS is the discovery of
tightly linked /functional markers
Novel approaches for BPH resistance

28. Schematic diagram of MAS and conventional breeding
(Jie Hu et al., 2016)

29. Chromosomal locations of BPH resistance genes/QTLs
along with markers used for MAS in rice
(Haliru et.al, 2020)

30. Contd.
(Haliru et.al, 2020)

31. derived lines through MAS
• Introgression line IR 71033-121-15 derived from the cross
between O.minuta and susceptible Korean japonica variety
”Junambyeo” carries two resistance genes Bph 20(t) and
Bph21(t). (Rahman et al.,2009)
• Introgression line IR 65482-7-216-1-2 derived from the cross
between O. australiensis and “Jinbubyeo “ carying Bph 18(t)
dominant gene. (Jena et al.,2006)
• Bph 14 and Bph15 transferred into the CMS rice restorer line in
China. The hybrid of Luohong4A/HB13002-50-9-7 had high
yield, BPH resistance, superior rice quality and desirable
agronomic performances. (Wang et al., 2016)

32. BPH resistance genes pyramided in rice
Pyramided genes/QTLs Trait Recipient background
Bph1+Bph 2 BPH resistance Tsukushibare
Bph6+Bph9 BPH resistance 93-11
Bph6+Bph12 BPH resistance 93-11, Nipponbare
Bph3+Bph27 BPH resistance 93-11,Ningjing
Bph14+Bph15 BPH resistance Minghui 63, Shanyou 63
Bph14+Bph 15 BPH resistance Huahui 938
Bph 14+Bph 15 BPH resistance Huang-Hua-Zhan
Bph3+Bph14+Bph15 BPH resistance Hemeizhan
Xa 21+Bph14+Bph15 BPH and BLB resistance Yuehui9113
Pita+Pi1+Pi2+xa5+Xa23
+Bph3
BPH, BLB and blast resistance HN88
Qbph12+sub1 BPH resistance and
submergence tolerance
KDML 105
Bph6+Bph9+Gn8.1+Rf3
+Rf4+Rf5+Rf6
BPH resistance, Big panicle,
fertility restoration
93-11
(Kumar et.al, 2020)

33. Novel approaches contd.
 Under the BPH infestation, expression of
 pathogenesis-related genes
 signalling molecule synthesis genes
 antioxident-related genes
 lignin biosynthesis-related genes
• Wound induced response genes activated after BPH attack
through signal(AOS-Allene oxide synthase )synthesis
activation. (Jannoey et al., 2017)
Increased
Gene expression

34. Model for rice resistance to BPH
(Jing et al.,2017)
PTI
(Pathogen
associated
molecular
pattern
triggered
immunity)
ETI
(Effector
triggered
immunity)
HAMPs
DAMPs Effectors
Lectin receptor
kinase protein
CC-NB-LRR
protein
immune response of rice to insect is regulated by hormone signaling pathways (JA,SA and ET)

35. Expression of BPH resistance genes
• Bph 14 gene encodes a coiled-coil nucleotide-binding and
leucine rich repeat (CC-NB-LRR) protein which resists BPH
attack at vegetative and maturity growth phases of rice
• This special LRR protein plays a role in sensing BPH attack
• It activates the host defense response mechanism, by
stimulating salicylic acid dependent resistance pathways
• It triggers the deposition of callose within the phloem tissue
which inhibits BPH feeding on the host plant (Du et.al, 2009)

36. • The Bph 29 encodes B3 DNA binding protein that activates
the salicilic acid signalling pathway and suppresses jasmonic
acid/ethylene dependent pathway
• It confers resistance by inducing callose deposition around
sieve tube in the phloem
• That inhibit the BPH feeding(Wang et al.,2015)
 The Bph3/Bph17 contains three duplicated genes encoding for
lectin receptor kinase. These are independently confer BPH
resistance and collectively function to confer broad spectrum,
durable resistant against BPH present in Rathu heenati (Liu et
al.,2015)

37. Transgenic approach for BPH resistance
• A snowdrop lectin gene(Galanthus nivalis agglutinin,GNA)
showed toxicity towards BPH when supplemented from an
artificial diet(Rao et.al.,1998)
• Transgenic indica rice(Chaitanya) plants containing the GNA
gene expressed resistance to BPH, driven by phloem specific
promoter(from rice sucrose synthase RSs 1 gene) and by a
constitutive promoter (From maize ubiquitin ubi1 gene)
• Insect bioassays and feeding studies showed that GNA gene
expressed in transgenic rice plant decreased survival and
production of off springs of the insect, retard insect
development and BPH feeding(Nagadhara et al.,2003)

38. • The transgenic plants expressing OsMKK3 , gene have high
level of jasmonic acid jasmonyl-L-isoleucine and abscisic acid
and reduced salicilic acid levels after BPH infestation
• This lowers the preference for feeding, oviposition, hatching rate
of eggs and nymphal survival rate (Zhou et al., 2019)

39. Genome editing
• In rice, the cytochrome P450 CYP71A1 (encoding tryptamine
5-hydroxylase) gene has been edited using CRISPR/Cas 9 for
the development of BPH resistant rice (Lu et al.,2018)
• The edited plants showed increased Salicylic acid levels and
decreased serotonin levels which leads to enhanced BPH
resistance

40. Allele mining
• Identification of new allelic variants of a gene from diverse
germplasm resources or distant wild relatives (Allele mining)
• 260 diverse germplasm collected from 32 countries have been
screened using eco-tilling (discover allelic variation present in
the natural germplasm resources) to identify the presence of
five BPH resistance genes (Bph10,13,18,20 and 21)
(Ramkumar et al.,2016)

41. Conclusion and perspective
• Enhancement of rice production faces serious threats due to frequent
population increase, consequences in climate change and evolution
of new BPH biotypes
• Use of resistant varieties is the most economically viable an
environmentally friendly strategy
• In recent years, marker aided gene pyramiding approach has become
prominent in BPH resistant variety development
• The majority of BPH resistant genes do not afford broad spectrum
resistant to various BPH populations
• Therefore it is essential to identify diverse genetic sources to
overcome resistance of new BPH biotypes

42. • Most of the markers, are not tightly linked to resistance genes
and therefore difficult to use as markers in practical breeding
for BPH resistance
• Accurate phenotyping, fine mapping of resistance gene loci,
and development of reliable PCR markers would be great
value in MAS
• Transgenic approaches for BPH resistance in rice are
successful, but its implementation faces regulatory issues,
political opposition in most countries

43. References
• Du, B., Zhang, W., Liu, B., Hu, J., Wei, Z., Shi, Z., … He, G. (2009).Identification and characterization of
Bph14, a gene conferring resistance to brown planthopper in rice. Proceedings of the National Academy of
Sciences of the United States of America, 106(52), 22163–22168. https://doi.org/10.1073/pnas.09121 39106
• Fernando, K.K.S, Ekanayake, E.M.D.S.B and Ketipearachchi, Y.(2007) Identification of biotypes of BPH
using microsatellite markers , Annals of the Sri Lanka Department of Agriculture, PP 27-33
• Haliru, B.S.,Rafii, M.Y.,Mazlan, N.,Ramlee, S.I.,Muhammad,I.,Akos, I.S.,Halidu, J.,Swaray, S. and Bashir,
Y.R, (2020), Recent strategies for detection and Improvement of Brown Planthopper resistance genes in
rice, Plant, 9,1202, https://doi.org/10.3390/plants9091202
• Hu, J., Xiao, C. and He, Y., (2016) Recent progress on the genetics and molecular breeding of brown
planthopper resistance in rice, Rice, 9:30, https://doi.org/10.1186/s12284-016-0099-0
• Jannoey, P., Channei, D., Kotcharerk, J., Pongprasert, W., & Nomura, M.(2017). Expression analysis of
genes related to rice resistance againstbrown planthopper, Nilaparvata lugens. Rice Science, 24, 163–
172.https://doi.org/10.1016/j.rsci.2016.10.001
• Jena, K. K., Jeung, J. U., Lee, J. H., Choi, H. C., & Brar, D. S. (2006). High resolution mapping of a new
brown planthopper (BPH) resistance gene, Bph18(t), and marker-assisted selection for BPH resistance in
rice (Oryza sativa L.). Theoretical and Applied Genetics, 112(2), 288– 297. https://doi.org/10.1007/s0012 2-
005-0127-8
• Jena, K. K., & Kim, S. M. (2010). Current status of brown planthopper (BPH) resistance and genetics. Rice,
3(2–3), 161–171. https://doi. org/10.1007/s1228 4-010-9050-y
• Kumar, K.,Kaur, P.,Kishor, A., Vikal, Y.,Singh, K. and Neelam, K., (2020). Recent advances in genomics-
assisted breeding of brown planthopper resistance in rice, Plant breeding.1052-1066,
https://doi.org/10.1111/pbr.12851
• Liu, Y., Wu, H., Chen, H., Liu, Y., He, J., Kang, H., … Wan, J. (2015). A gene cluster encoding lectin
receptor kinase confers broad-spectrum and durable insect resistance in rice. Nature Biotechnology, 33(3),
301– 305. https://doi.org/10.1038/nbt.3069

44. References contd.
• Lu, H. P., Luo, T., Fu, H. W., Wang, L., Tan, Y. Y., Huang, J. Z., … Shu, Q. Y.(2018). Resistance of rice to
insect pests mediated by suppression of serotonin biosynthesis. Nature Plants, 4, 338–344. https://doi.
org/10.1038/s4147 7-018-0152-7
• Nagadhara, D., Ramesh, S., Pasalu, I. C., Rao, Y. K., Krishnaiah, N. V., Sarma, N. P., … Rao, K. V. (2003).
Transgenic indica rice resistant to sap-sucking insects. Plant Biotechnology Journal, 1(3), 231–240.
https://doi.org/10.1046/j.1467-7652.2003.00022.x
• Nugaliyadda, L., Amarasinghe, A. A. and Hidaka, T. 2001, The rice brown plant hopper outbreak in
1997/1998 Maha season. Strategies to improve rice pest management in Sri Lanka. Annals of the Sri Lanka
Department of Agriculture, PP185-194.
• Rahman, M. L., Jiang, W., Chu, S. H., Qiao, Y., Ham, T. H., Woo, M. O., Koh, H. J. (2009). High-resolution
mapping of two rice brown planthopper resistance genes, Bph20(t) and Bph21(t), originating from Oryza
minuta. Theoretical and Applied Genetics, 119(7)1237–1246. https://doi.org/10.1007/s0012 2-009-1125-z
• Ramkumar, G., Prahalada, G. D., Hechanova, S. L., Kim, S. R., & Jena, K. K. (2016). Exploring genetic
diversity of rice cultivars for the presence of brown planthopper (BPH) resistance genes and development
of SNP marker for Bph18. Plant Breeding, 135(3), 301–308. https://doi. org/10.1111/pbr.12365
• Rao, K. V., Rathore, K. S., Hodges, T. K., Fu, X., Stoger, E., Sudhakar, D., Gatehouse, J. A. (1998).
Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper.
Plant Journal, 15(4), 469–477. https://doi. org/10.1046/j.1365-313X.1998.00226.x
• Tamura, Y.; Hattori, M.; Yoshioka, H.; Yoshioka, M.; Takahashi, A.; Wu, J.; Sentoku, N.; Yasui, H. Map-
based cloning and characterization of a brown planthopper resistance gene BPH26 from Oryza sativa L.
ssp. Indica cultivar ADR52. Sci. Rep. 2014, 4, 5872.
• Wang, Y., Cao, L., Zhang, Y., Cao, C., Liu, F., & Huang, F. (2015). Map based cloning and characterization
of BPH29, a B3 domain-containing recessive gene conferring brown planthopper resistance in rice. Journal
of Experimental Botany, 66(19), 6035–6045. https://doi. org/10.1093/jxb/erv318

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