Genetic and molecular_characterization_of_bph_tolerant_land_races_in_rice[1]

Acharya N G Ranga Agricultural University
Agricultural college, Bapatla
Course No : GP 591
Course Title : Masters Seminar
Topic : Genetic and Molecular Characterization of BPH Tolerant
Landraces in Rice
Submitted by
Y. Pravallika,
BAM-20-22 ,
M.Sc. (Ag) 1st year.
Submitted to
Dr. T. Srinivas,
Professor and Head,
Department of Genetics and Plant Breeding ,
Bapatla.

DEPARTMENT OF GENETICS AND PLANT BREEDING
AGRICULTURAL COLLEGE, BAPATLA
Contents:
• Rice introduction
• Morphology
• About Brown plant hopper (BPH)
• Screening techniques
• Biotypes and Resistant genes
• Case studies

Introduction :
• Rice is a self pollinated cereal crop having chromosome no 2n = 2x =24 belonging to
Gramineae family.
• It is the most important staple food for more than half of the worlds population.
• It is generally considered as an semi aquatic annual grass plant.
• It is a warm season crop, grown extensively in the humid and sub-tropical regions
of the world
• Rice is life as it is a prime source of energy rich carbohydrates in the diet.
• India ranks second in the production of rice after China.
• High yielding rice and wheat cultivars are generally led to birth of so called “ Green
Revolution “

Kingdom - Plantae
sub-kingdom - Tracheobionta
Division - Angiosperms
Class - Monocots
Subclass - Commelinidae
Order - Poales
Family - Poaceae
Subfamily - Bambusoidea
Genus - Oryza
Species - sativa
Origin - South East Asia
Botanical classification

• In the world, rice crop is cultivated in an area
of 162.06 M. ha and the production is 503.17
million metric tonnes (USDA 2020/2021).
• In India, area under rice is 44 M. ha and the
production is 121.46 million tonnes (USDA
2020/2021).
• In Andhra Pradesh, area under rice is 4.2 M.
ha and production of rice is 8658.8 thousand
tonnes (INDIASTAT 2020/2021).
Statistics

Nutrient content for 100g portion of Rice
• Rice is a good source of carbohydrates and
vitamin B and also provides smaller amounts of
minerals like iron and zinc etc.
Carbohydrates 26 g
Proteins 2.7 g
Fat 0.3 g
Dietary fiber 0.4 g
Potassium 35 mg
Sodium 1mg
Cholestrol 0 mg
Iron 10-11mg
Zinc 20 – 25 mg
Calcium 10mg
Magnesium 25 mg
Silicon 1.2 %

• Cultivated rice is an annual grass.
• Stem: It is made of nodes and internodes. Tillers
grow out of the main stem (culm).There are
primary, secondary and tertiary tilers.
• Roots : Fibrous root system consists of 2 types of
roots-seminal and adventitious roots.
• Seminal roots are temporary in nature and grows
out of radical.
• Leaves : They include the leaf blade and leaf
sheath. The upper most leaf below the panicle Is
‘Flag –leaf’.
Morphology Of rice :

• Inflorescence : Panicle that is
composed of spikelets. Spikelet consist
of 2 sterile Lemmas ,rachilla and the
floret. The rachilla is small axis b/w the
rudimentary glumes and the fertile
floret.
• Flower : Bisexual flower consisting of 6
stamens unlike 3 in the other cereals,
one pistil containing 1 ovule and the
perianth represented by ‘Lodicules’.
• Fruit : Caryopsis

• Landraces harbour a great genetic potential for rice improvement.
• Unlike high-yielding varieties (whose variability is limited due to
homozygosity), the landraces maintained by farmers are endowed with
tremendous genetic variability, as they are not subjected to subtle selection
over along period of time.
• This aids in the adaptation of landraces to wide agro-ecological niches and
they also have unmatched qualitative traits and medicinal properties.
• This rich variability of complex quantitative traits still remains unexploited.
• Landraces are also important genetic resources for resistance to pests and
fungal diseases. For instance, Indian landraces, Chemban and PTB33 is
resistant to BPH.

About Brown plant hopper (BPH) :
• 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
• However, the brown planthopper (BPH, Nilaparvata lugens ), 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).

Some Major Economic losses due to BPH
1. 1932 Japan famine – death of about 1 million people
• Due to severe attack of BPH on rice crop (Kaneda 1978)
2. 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.
3. 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)
4. 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.

Nature and Symptoms of damage :
• BPH is a small brown insect found mainly on the base of rice plants above the water level.
• Nymph and adult congregate at the base of plants, above water level, and suck plant sap.
• It is a typical vascular feeder primarily sucking phloem sap leading to hopperburn symptoms.
• In severe infestation the leaves first turn yellow, and later brown and finally the affected
plants dry and die.
• The first sign of damage is the sudden slumping of crop in patches in field.
• The crop in these affected patches dries up giving a scorched appearance called “Hopper
burn”.
• Crop loss is usually considerable and complete destruction of crop occurs in severe cases.
• The population growth of Brown plant hopper is maximum at a temperature range of 28 to 30
degrees and RH of 70 – 80 %.

Hopperburn symptom in rice field Life cycle of Brown planthopper

Score Symptoms Rating
0 No injury Immune
1 Slight yellowing of few plants Highly resistant (HR)
3 Leaves Partially yellow but with
no hopperburn
Resistant
5 Leaves with pronounced
yellowing and stunting or
wilting and 10-25 per cent of
plants with hopperburn,
remaining plants severely
stunted
Moderately resistant (MR)
7 More than half the plants with
wilting or with hopperburn,
remaining plants severely
stunted
Moderately susceptible (MS)
9 All plants dead Susceptible
BPH score will be
recorded using
the procedure
given by
Heinreichs et al.,
1985 on 0-9 scale

Screening techniques of BPH
1. Field screening
2. Standard seedbox technique
3. Field cage screening

Field screening :
• Using field populations – Used in early stages of plant
resistance programme
• Each genotype transplanted at 20×10 cm spacing in
two rows of one meter length.
• All around test entries, two meters of susceptible
variety were transplanted.
• Number of plant hoppers on 10 plants/entry were
recorded when susceptible check showed hopper
burn symptoms.
• Each entry was scored based on scoring system
developed by the International Rice Research Institute
• Populations may be either too
low or too high or unevenly
distributed in space or time
• Year-to-year variation in
population levels
• Field population may be
contaminated with non target
pest insect
Problems

Greenhouse Screening :
• This method involves growing of the test cultures in screening trays/seed boxes of size(50 X 40 X 7 cm).
• Fill the Seed boxes with well puddled and manure enriched soil and level.
• Draw 13 equidistant lines horizontally in the box.
• Draw two vertical lines in the centre of the box cutting the five lines on either side ofthe middle
horizontal line without touching the two boarder lines and middle horizontal fines.
• Soak the seed of test entries in the petridishes along with susceptible and resistant cheeks. Keep the
soaked seed in a plastic tray and cover with another tray.
• Next day,remove the water from the petridishes and allow entries to sprout.
• Sow 20 test entries in the test entry lines by using forceps.
• Sow two border rows with susceptible check, TNI and middle row with resistant check, PTB 33 for BPH.
• Sow at least 20 seeds of test entries per cach line and 40 seeds of susceptible and resistant checks per
line.
• This layout minimizes the chances of escape ofthe test entries from insect attack.
• Keep these seed boxes in big fibre trays in the plant growth chambers.

• 10 days (WBPH) - 12 days (BPH) after sowing when the plants are of 3-leaf stage, transfer these
seed boxes to the screening chambers and cover with cages made of mylar sheet.
• Release required number of first instar nymphs or the seedlings so that each seedling gets 6-8
nymphs.
• Cover these mylar cages with plastic mesh so that the insects cannot escape. This infestation is
sufficient to kill the susceptible check in 6-7 days.
• Monitor plant damage regularly.
• When TNI plants on one side show severe damage, rotate the tray by 180° for even reaction.
• When 90% of plants in the susceptible check, TNI on both sides are killed, the damage rating of
the entries is to be done.
• Score all the plants in a test entry and checks and score individually, total and average.
• Score the entries according to Standard Evaluation Scale (SES 2014) on 0-9 scale developed by
IRRI.

Field cage screening
• Limits emigration of the test insect
• Protection from predation and parasitism
Disadvantages-
• May cause abnormal environmental conditions
• Can alter plant growth, insect behavior
• Can cause foliar disease outbreak

Reason for Brown plant hopper Outbreak
• Continuous cultivation of susceptible varieties.
• Closer planting.
• Favorable microclimate
• Widespread migration of winged macropteras forms to endemic
and new areas.
• Continue uses of same group of insecticides (neonicotinoid) to be
avoided.
• Use of high doses of nitrogenous fertilizers.
• Longwinged planthoppers are being carried in the field by the
wind.

NEED FOR BPH RESISTANT VARIETIES IN RICE
• BPH is a monophagous pest causing yield loss upto 70%
• Chemical insecticides resulted in problems like toxicity to natural enemies
• Paddy ecosystem temperature range i.e. 28-30 degrees C is congenial for survival
and establishment of its threshold population
• Increased production cost of pesticides
• BPH sucks plant at the basal stem part, hence application of insecticides at
foliage is not effective
• Pesticide usage and resurgence of pest
• Long-term agro-ecosystem and human health damage by chemical insecticides

• Hence, rice resistance is a cost effective and environment
friendly strategy for BPH management
• This can be done by identifying new BPH resistant
germplasm
• Plant resistance to insect pests is categorized into 3 types:
a. Antibiosis b. Tolerance c. Antixenosis/Non preference.
• Recently the Bph 14 gene is cloned and found to encode a
protein with a leucine – rich – repeat domain similar to
those encoded by some pathogen resistant genes.
• This protein is probably involved in the direct or indirect
recognition of attack by planthoppers and activate salicylic
acid dependent pathway and resistance related
biochemical responses such as increased production of
trypsin proteinase inhibitor and callose deposition.
• These responses results in an antibiosis type resistance and
that dramatically reduces the survival, feeding and growth.

RELATION BETWEEN BIOTYPES OF BPH AND RESISTANCE GENES
• Biotypes are defined as a population or an individual distinguished from other
populations or individuals by non – morphological traits such as adaptation and
development in the particular host, host preference for feeding or oviposition
or both.
• 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.

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 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).

Mapped BPH resistance genes
• Thirty two 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.
• Among them 13 resistant genes are dominant and 8 are recessive.
• 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).

Management
1. ETL : ETL for Brown plant hopper 5 to 10 insect/hill.
2. Cultural control :
• Adopt planting with formation of alleys of 25 cm at intervals of 2mt to provide good aeration and
sunlight.
• Avoid dense planting
• Excess application of N fertilizer may be avoided.
• In vegetative phase of the crop growth periodical drying and wetting may be followed for short
period to create disturbance in micro climatic climate conditions favorable to pest development
• Grow resistance varieties like Chaitanya, Krishnaveni, Chandan, Triguna, Deepthi, Nandi, Vijeta,
Pratitha, Vajram etc.
• In developing resistant varieties bio type development has complicated the effectiveness
source resistant in one region may be susceptible in an other region.

Chemical control
• Application of these chemical insecticides were effective against
brown plant hopper in rice
1. Monocrotophos 36% SL 1.3ml/lit of water
2. Chlopyrifos 20 EC 2.5ml/lit of water
3. Carbaryl 50WP 2.0gm/lit of water
4. Phorate 10G@10kg/hac
5. Corbofuran 3G@25kg/hac
• The main method of controlling BPH is application of pesticides 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).

Evaluation of rice genotypes for brown planthopper (BPH)
resistance using molecular markers and phenotypic methods
Sai Harani et al., 2013
African Journal of Biotechnology
NAAS rating : 6.00
Case study - 1

Materials and Methods :
• The experimental material consisted of 28 elite rice genotypes available at
Barwale Foundation, viz; 1B, 2B, 7B, 8B, 9B, 14B, 16B, 18B, 21B, 22B, 24B, 25B,
28B,30B,36B,40B,41B,44B,IR129,1R150,IR157,IR168,Swarna,TN1,BPT5204,Pokkali,
and PTB33.
• TN1 and PTB33 were used as susceptible and resistant check, respectively

Standard seedbox screening technique
• The experiment was conducted at :
temp of 28 to 30 degrees, RH of 70 to 80 %
• The seeds were presoaked and sown in rows in 60 x 45 x 10 cm seed boxes
along with resistant and susceptible checks.
• 25 to 30 seedlings (Ten days old) per row per genotype were infested with
first instar nymphs at the rate of 8 to 10 nos. per seedling.
• Approximately one week after infestation “hopperburn” symptom was
observed.
• When more than 90% of susceptible check shows wilting, the plants were
scored individually based on scoring system proposed by the International
Rice Research Institute.

Genotype ID Code Score Genotype ID Code Score
IR58025 B 1B 8.3 IR73793 B 30B 6.9
IR62829 B 2B 7.3 IR68886 B 36B 5.8
IR68888 B 7B 5.5 IR79156 B 40B 5.6
IR68892 B 8B 6.1 IR80151 B 41B 5.8
IR68872 B 9B 7.9 IR80156 B 44B 5.6
IR69628 B 14 B 6 IR65482-7-2-16-1-2B IR129 4.32
IR70369 B 16 B 6.7 IR73680-4-5-10-2-1-2 IR150 4.3
IR70959 B 18 B 6.6 IR71033-121-15 IR157 4.7
IR72078 B 21 B 8.5 IR73885-1-4-3-2-10 IR168 5.8
IR72080 B 22 B 8 MTU 7029 Swarna 9
IR72018 B 24 B 6.6 Taichung native 1 TN1 9
IR73320 B 25 B 6.8 Samba Mahsuri BPT5204 9
IR73327 B 28 B 6.7 Traditional variety PTB33 1
IR73328 B 29 B 6.3 Landrace Pokkali 3
• Among all the 28 rice genotypes, PTB33 is widely used as donar parent for BPH by rice breeders consisting of bph2 and
bph3 genes and Pokkali which had bph9 gene, sacred as 1 and 3 respectively and TN1 showed a score of 9.

Nymphal survival method
• 20 newly hatched nymphs in a pot with three rice plants (40 days old) were placed inside the mylar cages
(45×5: H×R).
• The number of surviving nymphs was recorded every two days until they became adults (15 days).
• The experiment was carried in three replications and control plants were also maintained (Figure 3).
• All the plants were cut till the base of the stem and dried at 55°C for one week and biomass of infested
plants and control plants were weighed.
• Number of insects surviving on individual genotype was counted.

Figure7. SSR banding patterns of 28 rice genotypes from RM277 (A), RM3331 (B), RM510(C).

Conclusion :
• Standard seedbox screening technique – Among 28 rice genotypes, PTB33 is widely
used as donar parent consisting of bph2 and bph3 genes, Pokkali with bph9 gene.
• In case of honey dew test, genotypes showing high rate of phloem consumption are
usually susceptible and genotypes showing high xylem consumption are resistant
ones.
• In case of nymphal survival method, genotypes showing high no of genotypes with
less biomass content are susceptible and genotypes showing high biomass content
with less no of insects are resistant.
• In genotyping, out of the 34 markers used, four markers produced monomorphic
bands while 30 markers showed polymorphism by revealing 155 alleles.
• By this, we conclude that PTB33 and Pokkali is used as donar parents and TN1 is
used as susceptible check.

Screening of Rice Genotypes for Resistance to Brown Plant
Hopper Biotype 4 and Detection of BPH Resistance Genes
Durga Rani et al., 2015
Case study 2
International Journal of Life Sciences Biotechnology and Pharma Research
NAAS rating : 2. 53

• Plant material - The genotypes were obtained from the Andhra Pradesh Rice Research Institute and
Regional Agricultural Research Station (APRRI & RARS) Maruteru and Directorate of Rice Research
(DRR).
• Field screening - Each genotype was transplanted at 20×10 cm spacing in two rows of one meter
length. All around test entries, two meters of susceptible variety TN1 were transplanted. Scoring -
(0-9 scale).
• SSST – seeds sown in 60x45x10 cm seed boxes, 20-30 seedlings per genotype. Infestation @ 10
DAS. Scoring after one week when TN1 shows score 9.
• Total of 24 SSR markers were used for this study which are reported linked to 5 BPH resistant
genes.

Results :
• Out of 26 rice genotypes screened at APRRI, Maruteru, PTB33, BM71,
Rathuheenathi genotypes were rated as resistant (R), with average damage
score of 2, 2.5 and 3.0 respectively.
• Eleven genotypes viz., ACC5098, Deepthi (MTU4870), Bhavapuri Sannalu
(BPT1768), Akshaya (BPT2231), Vijetha (MTU1001), Cottondora Sannalu
(MTU1010), ACC2398, Swarnalatha, IR65482, Prabhath (MTU3626) and
MTU1064 showed moderate level of resistance with an average damage score
ranging between 3.5 and 6.0.
• Remaining varieties were susceptible showing damage score of >6.0

Conclusion :
• Although 32 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.
• From the above data, among the genotypes screened PTB33, BM71,
Rathuheenathi, ACC5098 and ACC2398 showed resistance towards BPH.
• Out of 24 SSR markers, only 2 markers shows the polymorphism. BM71 showed
donor allele with only one marker each, RM589 for Bph3 and RM3180 for Bph6
and ACC2398, ACC5098 (RM17008, RM3180), might be having Bph6 gene
• BM71 was hybridized with two mega rice varieties, Samba Mahsuri and Swarna to
combine BPH resistance with their high yield potential and superior grain quality.

Genetic Basis of Resistance to Brown Plant Hopper
(Nilaparvata lugensStal) in Local Landraces of Rice
Gangaraju et al., 2017
Case study 3
International Journal of Current Microbiology and Applied Sciences
NAAS Rating : 5.38

• The present study consisting experimental material resistant lines
(Ratnachoodi, Rajamudi and JBT 36/14)and susceptible lines (Jaya and TN-1).
• The brown planthopper population was maintained using standard artificial
rearing in the cages (plate 1).
• Study involving resistant X susceptible crosses a total of six F1were
generated from which F2 population was developed by selfing F1 plants.
• The F2populations of the crosses were evaluated for their reaction against
brownplanthopper in glasshouse conditions (Plate 2 & 3).
• Honey dew production of BPH on parents, F1 and F2 population of six
crosses

Results
Chi square test for goodness of fit F2 population Of the cross
between 3 resistant and 2 BPH susceptible parents

• Decreased amount of honeydew Excreted on Ratnachoodi, Rajamudi and JBT 3614 than the
susceptible checks Jaya and TN 1 and F1 population it was intermediate to the parents.
• Honeydew excreted on F2population was less as compared to the parents and F1
population.
• This clearly indicates that a major gene and number of minor genes are responsible for BPH
resistance
Conclusion :
• The F2’s of the crosses viz., Jaya × Rajamudi, Jaya × Ratnachoodi and TN 1 × JBT 3614
indicated that the resistance to BPH due to monogenic dominant gene action(Table 1) and
segregated in the ratioof 3:1, while, Jaya × JBT 3614 and TN 1 × Ratnachoodi segregated in
13:3, resistance is governed by two genes with inhibitory interaction and TN 1 × Rajamudi in
9:7 ratio with complementary gene action

Genetic variability, heritability, correlation and path
analyses of yield components in traditional rice
(Oryza sativa L.) landraces
Saha et al., 2019
Case study - 4
Journal of Bangladesh agricultural University
NAAS rating : *

• 40 rice landraces collected from Mymensingh and Sylhet divisions of
Bangladesh.
• The seed bed was prepared by raising the soil from the field surface to 5-10
cm above and then puddling.
• All the 40 genotypes were sown separately in seedbed.
• 30 days old seedlings were transplanted to the main plot with one seedling
per hill.
• Experimental design was Randomized Block Design with three replications.
• Plot size was 1m * 1m. Row to row and plant to plant distance was 20 *15cm.
• Data was recorded on 5 randomly chosen plants of each genotype for each
replication for the selected traits.

Results

Heritability
range (%)
Category Quantitative
traits
< 30% Low -
30-60 % Medium Days to maturity
> 60% High Remaining traits
GCV & PCV Category Quantitative
traits
< 10 % Low DF, DM, PL, PH
10 – 20 % Medium Pollen fertility
>20 % High Remaining traits
GA (% as mean) Category Quantitative
traits
< 10 % Low DM
10 – 20% Moderate DF, PH, PL
>20 % High Remaining traits
• Heritability range values was given by Johnson
et al., 1955
• GCV and PCV values was given by Subramanyam
et al., 1973
• Genetic advance (% as mean) values was given by Johnson et al., 1955

• In the present study residual
effect was 0.47 and 0.40 at
genotypic and phenotypic
level respectively.
• Residual effect (0.4) indicates
that the characters which were
selected in this study
contributed 60 % to the yield.
• Besides, some other factors like
sampling error, personal error
and geographical position which
have not been considered here
need to be included in this
analysis to account fully for the
variation in yield.

Conclusion :
• PCV was higher than the corresponding GCV for all the traits indicating that there was an
influence of the environment.
• The high estimates of PCV and GCV for these traits suggested the possibility of yield
improvement through selection of these traits.
• The high heritability values of the considered traits in the present study indicated that those
were less influenced by the environment and thus help in effective selection of the traits.
• High heritability along with high genetic advance was observed for the traits, viz., flag leaf area,
pollen fertility, number of grains per panicle path and number of filled grains per panicle
indicated that the characters are governed by the additive gene effects.
• Genotypic correlation coefficients in most cases were higher than their phenotypic correlation
coefficient indicating the association was largely due to the genetic reason.
• High direct effect along with positive and high indirect effects through other traits provide a
better chance for the character to be selected.

Genetic and molecular_characterization_of_bph_tolerant_land_races_in_rice[1]

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