SUBHASHREE PRIYADARSHINI, DEPT. OF ENTOMOLOGY, Ph.D IInd Year, ID No; RAD/2017-14, PROFESSOR JAYASHANKAR TELANGANA STATE AGRICULTURAL UNIVERSITY, HYDERABAD., TELANGANA, 500030

1. MOLECULAR APPROACHES IN RICE
PEST MANAGEMENT
PRESENTED BY
SUBHASHREE PRIYADARSHINI
RAD/17-14

2. CONTENTS
1. INTODUCTION
2. PEST SCENARIO OF RICE
3. MOLECULAR APPROACHES IN PEST MANAGEMENT
MARKER ASSISTED SELECTION
GENE PYRAMIDING
TRANSGENICS
RNAi TECHNIQUE
GENOME EDITING
4. FUTURE PROSPECTS
5.CONCLUSION

3. INTRODUCTION
 Most important food crops in the world
 Feeds more than half of the worlds population
 over 60 % of Chinese population (Muthayya et al., 2014).
In India, Production- 109.70 MT
Productivity- 2400 Kg/ ha (Indiastat, 2015- 16)
 Productivity affected by several biotic and abiotic factors
Insect pests cause 25 percent loss in rice (Dhaliwal and Arora, 1996)

4. MAJOR RICE GROWING AREAS IN WORLD
1. China,
2., India,
3.Indonesia,
4.Bangladesh,
5.Thailand,
6.Vietnam,
7.Burma,
8.Philippines,
9.Cambodia,
10.Pakistan
https://www.worldatlas.com

5. pink borer
Yellow stem borer
Rice gall midge
Brown planthopper
Green leaf hopper
Rice Hispa
PEST SCENARIO IN RICE

6. Rice leaf folder
Rice case worm
cutworm
Grass hopper
Swarming caterpilla
Root weevil
Root weevil
Whorl maggot

7. MOLECULAR APPROACHES
1.Marker Assisted Selection
2.Gene pyramiding
3.Transgenics
4.RNAi
5.Genome Editing

8. What is MAS
indirect selection process
 a trait of interest is selected based on a marker
 linked to a trait of interest .
Molecular Breeding Method- MAS- use of DNA markers
 that are tightly-linked to target loci
substitute for - phenotypic screening/selection.

9. • Marker Assisted Backcross (MABC)
• Marker Assisted Pyramiding
• Marker Assisted Recurrent Selection (MARS)
• Quantitative Trait Loci (QTL)
• Genomic Selection
Marker Assisted Selection (MAS)

10. Tagging and mapping resistance genes
molecular markers - major genes conferring resistance - BPH, WBPH,
GLH, have been tagged and mapped.
over 26 major resistance genes - BPH
 12 resistance genes - tagged and mapped.
 Bph1, bph2, Bph16 and Bph 18 are mapped on chromosome 12,
Bph14, Bph15 and Bph17 on chromosome 4,
Bph13 and Bph14 on chromosome 3,
Bph11 on chromosome 2
 Bph4 on chromosome 6.

11.  Bph10 and Bph18 are introgressed from O. australiensis,
Bph13 and Bph15 from O. officinalis,
Bph11 from O. eichingeri
Bph 14 from O. latifolia.
QTLs.
Wbph6 and two genes Wbph8, Wbph9 - from O. officinalis -
chromosome 11, 3 and 4, respectively.
 Additional gene like Ovc, determining ovicidal effects has been mapped
on chromosome 6.

12. GENE SOURCE
Bph1 Mudgo
bph2 ASD 7
Bph3, Bph17 Rathu Heenathi
Bph2 and Bph3 PTB 33
bph4 Babawee
Bph5 ARC 10550
bph6 Swarnalatha
Bph7 T 12
bph8 Chinnasahu
Bph9 Pokkali
bph10 O. australiensis

13. GENE SOURCE
Glh1 Pankhari 203
Glh2 ASD7
Glh3 IR8
glh4 Ptb8
Glh5 ASD8
Glh7 Maddai Karuppan
Glh8 DV 85
Glh9 IR 28
Glh10 IR 36
Glh11 IR2096-26-1-2
Glh12 ARC10313
Glh 13
Glh14
Asmaita
ARC11554
GLH

14. ZLH
Zlh1 Rathu Heenati
Zlh2 Ptb21
Zlh3 Ptb33
Gall midge
Gm1 Eswarakora
Gm2 Siam 29
gm3 RP2068-18-3-5
Gm4 Abhaya
Gm5 ARC5984
Gm6 Dukong 1
Gm7 RP2223
Gm8 Jhipiti
Gm9 Madhuri L9
Gm10 BG380-2
Gm11 MR1523
Source: Bentur et al., (2011); Fujita., (2010)

15. Marker-assisted selection for rice brown planthopper (Nilaparvata lugens)
resistance using linked SSR markers
Mahmoodreza et al., (2015)
28 polymorphic SSR markers
108 F3 progenies
cross of Rathu Heenati and MR276
against biotypes 2 and 3.

16. Conclusion:
•SSR markers RM545, RM401, RM22, RM5953, RM210, RM242, RM217, RM224 and RM1103
were significantly associated with BPH resistance to biotypes 2 and 3 of BPH in rice (P ≤ 0.01).
•These markers showed high selection accuracy for resistant plant sources with confirmation of
resistance effect.

17. MAS
ADVANTAGES
 Population size- less
 TOI can easily tressed –
molecular markers
 Less time required
LIMITATIONS
 Marker-Gene poorly
linked- wrong result
 Skillful approach
 Infrastructure

18. What is Gene Pyramiding ?
Watson & Singh (1953)
Definition- Gene pyramiding is defined as a method aimed at assembling
multiple desirable genes/QTLs from multiple parents into a single genotype
for specific/multiple trait through conventional breeding (Yunbi Xu, 2010)
A pyramid could be constructed with major genes, minor genes, defeated
genes, effective genes, ineffective genes, race-specific genes, non race-
specific genes or any other type of host gene that confers resistance.

19. 1. Enhancing trait performance by combining two or more complementary
genes.
2. Remedying deficits by introgression of genes from other sources.
3. Increasing the durability of insect resistance.
4. Broadening the genetic basis of released cultivars.
Objectives of gene pyramiding
Identification of resistant genes / gene sources
Transfer in to elite genotype deficient for that resistant gene
through BC
Fixation of that genotype
Strategy for gene pyramiding…

20. Pyramiding blast, bacterial blight and brown planthopper resistance
genes in rice restorer lines
Juan et al., (2016)
Parents for crosses Details
Rathu Heenathi (RH) Donor of Bph3
CBB23 Donor of Xa23
HN88 A restorer line containing Xa23 gene
Shuhui 162 A restorer line containing Pita gene
Zhongzu 14 A restorer line containing Pi1, Pi2 and xa5
genes with multiple resistance to diseases and
insects
•Ten new lines with blast, BB and/or BPH resistance genes were developed – MAS
technique .
•Only HR13 with resistance genes to blast, BB and BPH was obtained.
• four lines (HR39, HR41, HR42, HR43) - moderate resistance – BPH.

22. Pyramiding of two BPH resistance genes and Stv-bi gene using marker-
assisted selection in japonica rice
Xu et al., (2013)
•Two BPH resistance genes (Bph14 and Bph15) and one RSD resistance gene (Stv-bi ) were
successfully transferred into three japonica varieties via a marker-aided backcrossing
procedure.
•The progeny lines with Bph14 and Bph15 genes showed high resistance to BPH, while the
progeny lines with Stv-bi gene showed high resistance to RSD.

23. •The use of Bph14, Bph15 and Stv-bi greatly facilitates the development of rice varieties with
resistance to BPH and RSV.
•Gene pyramiding and molecular markers .

24. • Pyramiding Bph1 and bph2 into a japonica line
• PYL showed higher level of resistance =Bph1
Sharma et al., 2004
• Bph14 and Bph15 – hybrid rice breeding - in China
• PYL had higher resistance than SIL.
Li et al., 2006
• Used RDA (Representational difference analysis)
• OsBphi252- tightly linked to BPH resistance
Park et al.,2008
• Bph25 and Bph26 –East Asia
• PYL – best result
Fujita et al.,2009
• Bph14 and Bph15.
• seedling damage, antixenosis, honeydew
production
Hu et al., 2012
• Bph12 and Bph6
• PYL- lower nymphal survival, slower population
growth, caused less damage
Qiu et al., 2012

25. GENE PYRAMIDING
ADVANTAGES
 Multiple traits can be
transfered- single
background
 Durable resistance
LIMITATIONS
 Unrelated background
genes cant function
easily
 Antagonistic genes
 Evaluation is necessary

26. TRANSGENICS
Transgenesis - the process of introducing
an exogenous gene—
called a transgene—
into a living organism
so that the organism will exhibit a new property
and transmit that property to its offspring.
Transgenics- organism

27. Agrobacterium- mediated Gene Transfer
Particle Bombardment/ Biolistics
PEG (polyethylene glycol) Mediated transformation
Electroporation
Liposome fusion
Methods of transformation

28. Development of Indica Basmati rice harboring two insecticidal genes for
sustainable resistance against lepidopteran insects
(Riaz et al.,2006)
•Basmati 370
• a co-integrate vector pSM6 carrying cry1Ac and cry2A genes.
•The hygromycin resistance -selectable marker.
•biolistic gun.
•PCR, Dot blot, Southern blot, Western blot and ELISA
•Plants from five different transgenic lines exhibited 100% mortality
against rice leaf folder and segregated in Mendelian fashion.
• No synergistic or antagonistic effect of cry1Ac and cry2A genes on the
development of resistance against RFL and YSB.

29. Structure and partial restriction map of
plasmid pSM6. (B) Structure and partial
restriction map of plasmid pROB5.
Biotixicity of rice plants
of different stages
against yellow stem
borer.
T1– T3: four, three and
two months old
transgenic plants;
C1– C3: four, three and
two months old
untransformed plants.

30. •Plants from five different transgenic lines exhibited 100% mortality against rice
leaf folder and segregated in Mendelian fashion.

31. Transgenic Rice Plants Expressing a Fused Protein of Cry1Ab/ Vip3H Has
Resistance to Rice Stem Borers Under Laboratory and Field Conditions
(Chen et al., 2010)
Resistance evaluation of transgenic rice expressing Cry1Ab/Vip3H protein at different
developmental stage against C. suppressalis neonate larvae
•Six transgenic rice, Oryza sativa L.,
lines (G6H1, G6H2, G6H3, G6H4,
G6H5, and G6H6)
•expressing a fused Cry1Ab/Vip3H
protein,
•Asiatic rice borer, Chilo suppressalis
and the stem borer Sesamia inferens
•expression - Cry1Ab protein - main
stems and flagleaf.

32. Resistance evaluation of transgenic rice expressing Cry1Ab/Vip3H protein
at different developmental stages against S. inferens neonate larvae

33. Laboratory evaluation of transgenic Bt rice resistance against rice leaf
roller, Cnaphalocrocis medinalis
(Kim et al., 2017)
8 transgenic Bt rice events
 with a synthetic cry1Ac gene
Neonates and third instar larvae
of Cnaphalocrocis medinalis,
Neonate larva- feeding avoidance
and death by starvation on six Bt
rice events.
 third instar larvae however, only
two events resulted in feeding
avoidance.

34. Feeding behavior of neonates of
C. medinalis fed on control
non-Bt rice (A) and T3
generation Bt rice 608103 (B).
Arrow heads indicate feeding
area of C. medinalis larvae.
Feeding behavior of 3rd instar
larvae of C. medinalis fed on
control non-Bt rice (A) and T3
generation Bt rice 608103 (B).
Arrow heads indicate feeding area
of C. Medinalis larvae.

35.  Cowpea trypsin inhibitor (CpTI) was introduced into rice.
 high-level accumulation of the CpTI protein in transgenic rice plants.
 Two species of rice stem borers.
Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic
rice plants confers resistance to two major rice insect pests
Xu et al., (2004)
Resistance to green leafhopper (Nephotettix virescens) and brown planthopper
(Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus
nivalis agglutinin; GNA)
Foissac et al., (2000) Survival was reduced
GNA binding to glycoproteins
BPH contained more “receptors” than GLH,
the binding affinity was stronger, particularly in the midgut.

36. TRANSGENICS
ADVANTAGES
 Novel trait- unrelated
organism
 Efficient method
LIMITATIONS
 Unpredicted result
 GMO- Not acceptable in
India
 Skillful approach
 Laboratory set up

37. RNAi TECHNIQUE
Highly conserved, sequence-specific mechanism
triggered by the presence of double-stranded RNA (dsRNA)
COMPONENTS OF RNAi
 ds RNA : Longer than 30 nt
 DICER:
Involved in the initiation of RNAi.
Digests dsRNA into uniformly sized siRNA.
ATP-dependent nucleases
 Si RNA: 21-25 nt
 RISC: (RNA-induced silencing complex):
RISC is a large (~500-kDa) RNA-multi protein complex Argonaute proteins cleave
the target mRNA strand.

39. Shut down proteins related to metabolism or reproduction
Highly conserved system : RNAi core components
 Specificity to target the gene: Sequence specific
 Stability: RNAi Effects are stable
 Targeting multi-genes: Multi genes with similar sequences
 Wide adaptability: Seven different orders
 Systemic and heritable: Signal spread; Dominant
 Alternate approach for insect resistant transgenics

40. Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated
RNAi in Nilaparvata lugens
- Zha et al. (2011)
Hexose transporter gene -
NlHT1 -
Carboxypeptidase gene -
Nlcar
Trypsin-like serine protease
gene - Nltry
NlHT1, Nlcar and Nltry suppression in
nymphs fed on transgenic plants

41. A
a1
a2
B
C
c1
c2
Photograph comparing Rice stripe virus (RSV) resistance of T0, T1 and T5 generation transgenic seedlings with the
control. A, typical disease symptoms in infected plants (a1, wild type (WT) plant; a2, susceptible control). B, T0 of
transgenic seedlings (b1 is WT plant, the others are transgenic seedlings categorized as class 0 or 1). C, T2 of
transgenic seedlings (c1, transgenic line 969B-1; c2, WT plant). D, T5 of transgenic seedlings (d1, Zhendao 88; d2,
transgenic line 969B-1-2-1-1; d3, susceptible control; d4, WT seedlings).
RNAi-mediated transgenic rice resistance to Rice stripe virus
(Li et al., 2016)
•Three RSV-resistant transgenic rice lines
•targets - RSV nucleocapsid protein gene
•expression of siRNAs

42. New innovations in agricultural biotech: Consumer acceptance of topical
RNAi in rice production
(Shew et al., 2017)
survey in the USA, Canada, Australia, France, and Belgium
CONCLUSION
 consumers in the USA, Canada, Australia, and France still require a discount for rice
produced with topical RNAi compared to conventionally-produced rice .
 consumers in all countries were more willing to consume rice produced with non-
GM RNAi than with GM Bt technology .
 These findings suggest consumers differentiate among biotechnology solutions and
consumers may prefer topical RNAi insect control to transgenic GMO insecticides.

43. Silencing of CYP6 and APN Genes Affects the Growth and Development of
Rice Yellow Stem Borer, Scirpophaga incertulas
Kola et al., (2016)
A) Schematic representation of dsRNA labeled with 5FAM, Fluorescence detection of
dsRNAi nTN1 cutstems under microscope
(B) Untreated
(C) Treated cutstem.
Cytochrome P450 monooxygenases (CYP6):
•Hormonal regulation,
•metabolism of xenobiotics
•Biosynthesis of endogenous compounds
Aminopeptidase N (APNs):
•dietary protein digestion majorly located
at the midgut epithelium.

44. (A) Effect of CYP6 dsRNA on YSB larvae fed on treated TN1 cut stems ,Growth reduction in treated larvae
at (a) 6thDAT, (c) 12thDAT (e) 15thDAT after feeding on dsRNA treated TN1 cut stems. Presence of
fluorescence in larval midgut indicating that dsRNA was ingested by larvae when fed on treated stems at
(b) 6thDAT, (d) 12thDAT, (f) 15thDAT.
(B) Effectof Amino dsRNA on YSB larvae fed on treated TN1 cutstems. Growth reduction in treated larvae
at (a) 6thDAT, (c) 12thDAT (e) 15th DAT after feeding on dsRNA treated TN1 cutstems. Presence of
fluorescence in larval midgutat (b) 6th DAT, (d) 12th DAT, (f) 15th DAT.

45. Relative expression fed on TN1 cut stems treated with CYP6-dsRNA and APN-dsRNA
in an individual bioassays. b-actin and 18s were used as an internal controls (A)
expression of CYP6 gene from larvae sample dat 6, 12, 15 days after treatment along
with respective controls. (B) Expression of APN in 6, 12, 15 days after treatment along
with respective controls.

46. PSB larvae fed on rice TN1 cutstems, 6th DAT (A) Control, (B) APN treated,
(C) CYP treated.12thDAT (D) Control, (E) APN treated, (F) CYP
treated.15thDAT (G) Control, (H) APN treated, (I) CYP treated.

47. RNAi
ADVANTAGES
 Precise silencing
 Without affecting other genes
 Natural occuring phenomena
 Easy to employ
LIMITATIONS
 Only Knock down effect
 No knock out
 Off target effect

48.  Recent technology,
Target site modification
Increasing the precision of the correction or insertion
Preventing any cell toxicity
GENOME EDITING

49. DNA Double Strand break RepairGeneral
principles
The genome of a cell is continuously damaged.
By-products of the cell's own metabolism
 such as reactive oxygen species
can damage DNA bases and
 cause lesions
that can block progression of replication.
The result is double-strand breaks (DSBs) in
the chromosome.
Also caused by environmental exposure
 to irradiation,
other chemical agents,
 ultraviolet light (UV).

50. GENOME EDITING
TECHNIQUES
1. MEGANUCLEASES
2. ZINC FINGER NUCLEASES
3. TRANSCRIPTION ACTIVATOR LIKE EFFECTOR NUCLEASES
4. CLUSTERED REGULARLY INTERSPACED SHORT
PALINDROMIC REPEATS

51. • Clustered Regularly Interspaced Short Palindromic Repeats
• Bacteria use as a kind of acquired immunity to protect against
viruses
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS

53. Spotted wing drosophila
adult flies (A), genitalia (B)
and dissected reproductive
tissue (C) of wild type
female, male (WT), and
homozygous mutants (M)
for the CRISPR/Cas9
interrupted sex lethal (Sxl)
gene. Note that the
mutants developed an
intersexual phenotype with
abnormal genitalia and
abnormal ovaries.
(Graphic: Adapted from Li
and Scott 2016 Biochem
Biophys Res Commun)

54. ADVANTAGES OF CRISPR
 simplicity and efficiency.
 Since it can be applied directly in embryo, CRISPR/Cas9 reduces the time
required to modify target genes compared to gene targeting technologies based
on the use of embryonic stem (ES) cells.
 Improved bioinformatics tools — to identify the most appropriate sequences to
design guide RNAs .
 optimization of the experimental conditions enabled very robust procedures
which guarantee successful introduction of the desired mutation.

55. FUTURE PROSPECTS
In India , seven of the gall midge resistant gene markers have been validated in
alternative mapping populations and can be used in MAS.
However, markers for BPH need to be validated for Indian donors and used.
 Markers need to be developed for QTLs associated with resistance against
Indian populations of the pest.
Resistance genes for Yellow stem borer need to be tagged and markers
developed.

56. LITERATURE CITED:
Chen, Y., Tian, J. C., Shen, Z. C., Peng, Y. F., Hu, C., Guo, Y. Y. and Ye, G. Y. 2010. Transgenic
Rice Plants Expressing a Fused Protein of Cry1Ab/ Vip3H Has Resistance to Rice Stem Borers Under
Laboratory and Field Conditions. Journal of Economic Entomology. 103(4):1444-1453.
Dhaliwal, G.S. and Arora, R. 1996. Principles of insect management. Commonwealth Publishers, New
Delhi.
Juan, J. Z., Shudon, Y., Yuxiang, Z. , Yan, L., Changdeng, Y. and Qian, Q. 2016. Pyramiding blast,
bacterial blight and brown planthopper resistance genes in rice restorer lines. Journal of Integrative
Agriculture. 15(7): 1432–1440.
Khan, Z.R., Litsinger, J.A., Barrion, A.T., Villanueva, F.F.D., Fernandez, N.J. and Taylor, L.D., 1991.
World bibliography of rice stem borers, IRRI LosBanos, Philippines, pp. 1794– 1990.
Kim, J.H., Lee, S.Y., Choi, J.Y., Fang, Y., Ha, K.B., Park, D.H., Park, M.G., Woo, R.M., Kim, W.J.,
Kim, J.K. and Je, Y.H. 2017. Laboratory evaluation of transgenic Bt rice resistance against rice leaf
roller, Cnaphalocrocis medinalis. Journal of Asia-Pacific Entomology .20 : 221–224.
Kola, V.S.R., Renuka, P., Padmakumari, A.P., Mangrauthia, S.K., Balachandran, S.M., Ravindra Babu,
V. and. Madhav, M. S. 2016. Silencing of CYP6 and APN Genes Affects the Growth and Development
of Rice Yellow Stem Borer, Scirpophaga incertulas. Frontiers in Physiology . 7: 20.

57. Li, L., Cheng, G., Biao, W., Tong, Z., Yang, L., Yuhua, D., Wen, H., Chun, L. and Xifeng, W. 2016. RNAi-
mediated transgenic rice resistance to Rice stripe virus. Journal of Integrative Agriculture . 15(11):
2539–2549.
Mahmoodreza, S., Yusop, M. R., Ashkani, S., Musa, M. H., Adam, N. A., Haifa, I., Harun, A. R.,
and Latif, M. A. 2015. Marker-assisted selection for rice brown planthopper (Nilaparvata lugens)
resistance using linked SSR markers. Turkish Journal of Biology. 39: 666-673.
Riaz, N., Husnain, T., Fatima, T., Makhdoom, R., Bashir, K., Masson , L.,Altosaar, I. and Riazuddin, S.
2006. Development of Indica Basmati rice harboring two insecticidal genes for sustainable resistance
against lepidopteran insects. South African Journal of Botany. 72 : 217 – 223.
Shew, A.M., Danforth, D. M., Nalley, L. L., Nayga, R.M., Tsiboe, F. and Dixon, B. L. 2017. New
innovations in agricultural biotech: Consumer acceptance of topical RNAi in rice production. Food
Control . 81: 189-195.
Singh, B., Arora, R. and Gosal, S.S. Biological and Molecular Approaches in Pest Management. Scientific
Publishers, Jodhpur.
Xu, J. 2013. Pyramiding of two BPH resistance genes and Stv-bi gene using marker-assisted selection in
japonica rice. Crop Breeding and Applied Biotechnology . 13: 99-106.
Zha W., Peng X., Chen R., Du B., Zhu L and He G. 2011. Knockdown of midgut genes by dsRNA-transgenic
plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens. PLoS ONE. 6(5): e20504.