MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF GENOTYPES DERIVED FROM WILD CROSSES OF PADDY.

1
G.Mallikarjuna,
BAM-19-13,
M.Sc (Ag.,) 2nd Year,
Department of GPBR.
WELCOME

ACHARYA N.G RANGAAGRICULTURAL UNIVERSITY,
AGRICULTURAL COLLEGE,BAPATLA.
MASTER'S SEMINAR
TOPIC : Morphological and molecular characterization of genotypes derrived from
wild crosses of paddy.
COURSE NO : GP-591
DEPARTMENT : Genetics and Plant Breeding
SUBMITTED TO : SUBMITTED BY:
Dr .T.Srinivas, G. Subash Chandra Bose,
Professor and Head , BAM/20-26,
Department of GPBR . Department of GPBR.
2
Agricultural College, Bapatla
Department of Genetics and Plant Breeding 1

3
Department of Genetics and Plant Breeding
Morphological and molecular characterisation of genotypes
derived from wild crosses of paddy.
2

4
INTRODUCTION
BOTANY OF RICE
IN TO THE WILD SPS
VARIABILITY
CONTENTS
HERITABILTY
CORRELATION
GENETIC ADVANCE
CASE STUDIES
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TAXONOMIC POSITION
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• Kingdom :Plantae
• Sub-kingdom :Tracheobionta
• Division : Angiosperms
• Class :Liliopsida
• Subclass :Commelinidae
• Order :Cyperales
• Family :Poaceae
• Genus :Oryza
• Species :sativa

is the world’s most important cereal crop which
serves as a staple food for over 60% of the world’s population with an enormous
nutritional and economic impact (Singh and Singh, 2008).
 Approximately 90% of rice grown on the planet is produced and consumed in
Asia (Singh et al., 2015) .
The cultivation of rice is more than any other crop in the world with cultivated area of
162.57 million hectares with production of 503.17 million metric tonnes (USDA,
2020-2021) .
Among rice growing countries, India has largest area under rice cultivation in the
world i.e. 43.86 million hectares and ranks second in the production with 99.24 million
tonnes and the productivity of 2.49 t/ha next to wheat. (Ministry of Agriculture,
Government of India, 2018-19).
India is the second largest producer of rice after China occupying 43.8 million hectare
area with an average grain yield of 3.99 metric tonnes per hectare while the total
production is 116.48 million metric tonnes in 2018-19 (USDA, 2020) .
6

7
In A.P In India
22.08 lakh ha 43.86 million ha
123.52 Lakh
tonnes
99.24 million
tonnes
5.59 tonnes /
ha
2.49 t/ha
6
AREA
PRODUCTION
PRODUCTIVITY

Self-pollinated crop
Semi-aquatic plant and consists of arenchymatic
tissues. The presence of arenchymatic cells on
leaf, culm and roots can diffuse oxygen from
aerial parts downward to roots.
Root System:
The root system is fibrous.
The real functional roots are secondary
adventitious roots that are produced from the
lower nodes of the culm.
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BOTANY OF RICE

• Shoot System:
The rice stem known as culm is hollow and is made up of nodes
and internodes. Each node bears a leaf and bud, which may grow
into a shoot or tiller. Primary tillers grow out of the main culm.
Tillering continues in rice upto vegetative phase.
Some tillers die during the reproductive phase due to competition
for water and nutrients. Panicles bearing tillers are known as
fertile or productive tillers.
• Leaf:
Each node of the culm bears a leaf. Each leaf consists of the
following parts:
Leaf sheath, leaf blade, auricles and ligules.
Flag leaf: It is the uppermost leaf just below the panicle. It is
generally shorter in length and remains erect at an angle
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• Panicle:
The inflorescence of rice plant is born on terminal shoot and is known as
panicle. It is determinate type and at maturity it is droopy in nature.
Panicle bears the spikelets.
• Spikelet: A spkelet is the floral unit and consists of two sterile lemmas, a
lemma, a palea and the flower.
• Flower: It consists of 6 stamens with two -celled anthers and a pistle with
one overy and two stigmas. The pistil consists of one ovule.
• Grain:
Rice grain is the ripened ovary with lemma and palea firmly adhered to it.
The rice fruit is a caryopsis in which single seed is fused with the wall of
the ovary (paricarp).
The seed consists of endosperm and an embryo. The embryo is very small
and is found on the ventral side of the caryopsis. It contains plumule
(embryonic leaves) and radicle (root).
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Global food demand is expected to nearly double by 2050 due to an increase in the world’s
population.
The Green Revolution has played a key role in the past century by increasing agricultural
productivity worldwide, however, limited availability and continued depletion of natural resources
such as arable land and water will continue to pose a serious challenge for global food security in
the coming decades.
High yielding varieties with proven tolerance to biotic and abiotic stresses, superior nutritional
profiles, and the ability to adapt to the changing environment are needed for continued
agricultural sustainability.
 Part of the solution to increasing food production on the same or less cultivated land lies in
exploiting the subset of genes lost during the domestication process and subsequent targeted
breeding.
The narrow genetic base of modern cultivars is becoming a major bottleneck for crop
improvement efforts and, therefore, the use of crop wild relatives(CWRs) is a promising approach
to enhance genetic diversity of cultivated crops.
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Department of Genetics and Plant Breeding `10
WILD RICE SPECIES

Insect Tolerance:-
The brown planthopper (BPH), Nilaparvata lugens Stål, is a migratory
insect that has become the most devastating pest of rice.
 In addition to causing severe plant damage resulting in significant
production losses, BPH also transmits two disease causing viruses, rice
grassy stunt virus and rice ragged stunt virus.
Wild species of rice like O.officinalis(bph11(t), bph12(t), Bph13(t),
Bph14, and Bph15), O.rufipogon (bph29 and Bph30) are an important
source of brown plant hopper resistance genes(Insect Tolerance) .
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Tolerance to Biotic Stresses

• Disease Tolerance :-
Rice blast is considered the most serious and economically important
disease caused by a fungal pathogen Magnaporthe oryzae .
Wild species of rice have been found to harbor resistance genes to
rice blast (O. minuta, O. rufipogon ( R gene clusters (Piz, Pik, and
Pita), Bacterial leaf blight (O. rufipogon(Xa23), O. minuta (Xa27), O.
officinalis (Xa29(t)), and Rice
tungro bacilliform virus(O. longistaminata ,O. rufipogon ).
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• Drought and Heat Tolerance :-
Drought is one of the main environmental stressors that reduces agricultural
productivity in rice.
 Although low yielding, O. glaberrima, has been found to be an excellent source
of tolerance for drought.
• Acid Soil and Aluminum Tolerance:-
Aluminum toxicity is of utmost concern when rice is grown in acidic soils since it
adversely affects root development, water and nutrient uptake, and growth
resulting yield loss.
Wild species of rice, O. rufipogan, shows tolerance to the Acid soils,aluminium
toxicity, cold and salinity (O.coarctata).
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Tolerance to Abiotic Stresses

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Presence of differences among the individuals of plant population .
Due to differences in genetic constitution .
Due to differences in environment.
Essential for resistance to biotic and abiotic factors and adaptability
• Estimation of genotypic and phenotypic variances :
 Genetic variability forms the basic factor to be considered while making selection
Genotypic and phenotypic variances were estimated according to the formula given by Johnson et al.
(1955).
Where, GMS = genotypic mean square; EMS = error mean square and r = number of
replication
• Phenotypic variance :
Where, σ2g = Genotypic variance
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σ2p = σ2g + EMS
Variability

Heritability indicates transmissibility of a character in future generations
(Satheeshkumar and Saravanan, 2012).
High heritability coupled with high genetic advance is more helpful in forecasting
genetic gain (Johnson et al. 1955).
Heritability in broad sense (h2b) was estimated according to the formula
suggested by Johnson et al. (1955).
 Where, h2b=Heritability in broad sense
Heritability was classified as low (below 30%), medium (30- 60%) and high (above 60%) as
suggested by Johnson et al. (1955).
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Heritability

 Selection based on only yield is often unwise. So it is necessary to know the
association between yield and yield components which is determined by
correlation analysis (Akhtar et al. 2011).
It is a statistical measure which is used to find out the degree (strength) and
direction of relationship between two or more variables.
It determines the component characters on which selection can be based for
genetic improvement in yield.
The association of one or more characters influenced by a large number of genes
is elaborated statistically by correlation coefficients.
Genotypic correlation coefficient provides a measure of genotypes conjugation
between characters
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Correlation coefficient

Estimation of genotypic coefficient of variation (GCV) and phenotypic coefficient
of variation (PCV) GCV and PCV values were estimated according to the formula
given by Burton and De Vane (1953) and Singh and Chaudhury (1985).
Genotypic coefficient of variation :
Where, x = Population mean
 Phenotypic coefficient of variation :
GCV and PCV values were categorized as low (<10%), moderate (10-20%) and
high (>20%) (Sivasubramanianand Madhavamenon, 1973)
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Genetic advance :
Genetic advance or response to selection is a measure of how much gain you
may get from phenotypic selection for a trait.
It mainly depends on selection intensity I (i=2.06 at 5% level), broad sense
heritability (h2) of character and phenotypic standard deviation of trait (measure of
phenotypic variability).
Genetic advance was estimated following the formula given by Johnson et al.
(1955).
Where, K= Selection differential, the value of which is 2.06 at 5% selection
intensity ; σp = Phenotypic standard deviation
20
GA = h2b.K.σp

21

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case study :1
NAAS Rating (2021): 5.14

107 elite rice genotypes developed at Regional Agricultural Research Station
(RARS), Maruteru and Agricultural Research Station (ARS), Bapatla.
Sown during Kharif 2017 at RARS , Maruteru in a randomized block design with
two replications.
For transplanting, nursery was raised separately and 28 days old seedlings were
transplanted in the main field with a spacing of 20×15 cm.
• Observations were recorded on five randomly selected plants for grain yield per
plant (g) and yield component characters namely : days to 50% flowering, plant
height (cm), the number of ear bearing tillers per plant, panicle length (cm), the
total number of grains per panicle, spikelet fertility (%) and test weight (g).
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MATERIALAND METHODS

 High GCV and PCV coupled with a high heritability and high genetic advance as per
cent of mean was observed for the number of ear bearing tillers per plant indicating
the pre-ponderance of an additive gene action and therefore the scope for
improvement of the trait through selection.
Heritability estimates for the various traits studied ranged from 60.39 (grain yield
per plant) to (98.00) days to 50% flowering. High estimates of heritability (> 60%)
were recorded for all the traits studied.
 High genetic advance as per cent of mean was recorded for plant height, the
number of ear bearing tillers per plant, the total number of grains per panicle, test
weight and grain yield per plant.
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RESULTS AND DISCUSSION

25

These results revealed a positive and significant association of grain yield with
days to 50% flowering, plant height, the number of ear bearing tillers per plant,
panicle length, the total number of grains per panicle, spikelet fertility and test
weight.
26

Analysis of the direct and indirect effects also revealed a high (>0.3) positive direct effect for the
number of ear bearing tillers per plant (0.4202), the total number of grains per panicle (0.3279) and
test weight (0.3699), in addition to significant and positive association with grain yield per plant.
High direct effects of the traits therefore appear to be the main factor for their association with grain
yield per plant.
27

 Ear bearing tillers per plant had recorded a high variability,
heritability and genetic advance as per cent of mean in addition to
correlation and direct effects with grain yield per plant indicating its
effectiveness as important selection criterion for the yield
improvement.
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CONCLUSION

29
case study :2
Journal of Agriculture and Natural Resources NAAS Rating (2021):-##--

Conducted at Regional Agricultural Research Station, Dipayal, Doti.
Twenty six rice genotypes received from National Rice Research Program, Hardinath was
experimented on randomized complete block design in 3 replications during rainy season of
2015.
Spacing :25 cm and 15. Individual plot size was 6 m2.
Ten plants from middle row of each plot were randomly selected and plant height, panicle
length, fertile grains per panicle were taken.
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MATERIALS AND METHOD

Analysis of variance revealed the existence of significant difference for days to flowering,
maturity, plant height, panicle length, thousand grain weight and grain yield.
 High heritability was estimated for days to flowering (0.88), maturity (0.79), thousand
grain weight (0.48) and plant height (0.43) suggesting these traits are under high genetic
control.
High phenotypic variation was observed for grain yield (24.87%), number of grains/panicle
(22.45%), number of panicles/m2 (20.95%) and straw yield (20.75%) while grain yield had
medium (12.02%) and remaining traits showed low genotypic coefficient of variation
(<10%).
High phenotypic coefficient of variation estimated as compared to genotypic coefficient of
variation showed environmental influence on the expression of traits.
Grain yield (11.98) and days to flowering (10.32) showed medium and remaining traits
showed low genotypic advance as percent of mean.
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RESULTS AND DISCUSSION

32
High to low heritability with moderate to low genotypic advance as percent of mean suggested
these traits were governed by non additive gene thus direct selection is not beneficial. Further
improvements on yield potentiality and yield traits on these genotypes are suggested by creating
variation and selection.

33
 Panicle length (r = 0.230), days to flowering (r = 0.247), effective tillers (r = 0.488)
and straw yield (r = 0.846) manifested significant positive association with grain
yield indicating that yield can be increased if selection applied in favor of those yield
components.

This study generally indicated that there was genetic variability among the
genotypes studied mostly inheritance by non additive gene action, hence direct
selection is not fruitful on these populations.
Thus hybridization, mutation breeding then selection and progeny testing
methods is recommended for improvement on traits of interest.
Selection applied in favour of days to flowering, effective tillers, panicle length
and straw yield will chance yield potential of rice genotypes as these traits
manifested significant positive association with grain yield.
 On the basis of mean performance, high grain yield, maturity, thousand grain
weight were exhibited by the genotypes IR09F4-36, IR88965-19-3-1-2 and
IR79906-192-2-3 which were found suitable genotypes for cultivation in rainfed
rice wheat system.
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CONCLUSION

35
case study :3
NAAS Rating (2021):-##--

Mature seeds of rice cultivars BR3, BR4, BR11, BRRI dhan28, BRRI dhan29, BRRI
dhan34, and BRRI dhan37 were collected from regional station of Bangladesh
Rice Research Institute (BRRI, Rajshahi).
randomized block design with three replications in 2 m2 plot.
In every plot 10 rows each containing 10 single seedlings of all the cultivars were
transplanted.
 15 and 30 cm.
At maturity 10 guarded plants were randomly selected from each replication and
data were collected for plant height, flag leaf length, days to heading, panicle
length, grain per panicle and other yield contributing characters including grain
weight, and grain yield per plant at harvest.
36
Materials and Methods

Flag leaf length positively correlates with yield.
 Length of flag leaf and panicle of two rice cultivars, BR11 and BRRI dhan28 were measured and
correlation between the characters was calculated.
The correlation between FL and yield was positive and highly significant.
37
Results and Discussion

38
B
A

39

40

In rice, the flag leaf is metabolically active and critically important in determining
yield.
It has been assigned an important role in terms of supply of photosynthates to
the grains.
Any damage done to that leaf will have a direct and dramatic impact on crops
potential.
It is important to protect the flag leaf in the early stages of crop.
 Raising the photosynthetic capacity of flag leaf is the key to overcome the
photosynthate-source restriction on grain yield and to make a new breakthrough
of yield potential in future development of rice.
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CONCLUSION

42
CASE STUDY : 4

43
Advances in rice breeding it is essential to understand the relatedness and ancestry of
introduced rice accessions and identify SSR markers associated with agronomically
important phenotypic traits, for example yield.
Identification of a candidate gene for panicle length in rice through association
mapping is an important trait for improving panicle architecture and grain yield in
rice.
Association mapping is one of the feasible options to identify major effect QTLs for
yield traits in rice.
 An association mapping for12 agronomic traits was carried out using a core
collection of rice consisting of 100 landraces (Panel 1) with 44 simple sequence
repeat (SSR) markers.
Introduction

100 genotypes of rice were taken for this research.
All of the germplasm were cultivated at the instructional farm of Indira Gandhi Agricultural University,
Raipur, in wet season (Kharif) 2018.
 21 days old seedlings were transplanted in the field.
 25 cm and 15 cm.
Thirty plants of each variety were grown in three rows with 10 plants per row.
 For each block, the five plants in the middle position of the second row of each variety were selected
so that the marginal effect was avoided.
A randomized complete block design with three replications.
Heading date (HD),plant height (PH), panicle length (PL), flag leaf length (FLL), and flag leaf width (FLW)
were measured in centimetres. Number of effective tillers (NT) was counted as effective tillers,1000-
grain weight (1000GW), Biological yield (BY) and Harvest index (HI).
44
Materials and Methods

45

44 SSR markers evenly distributed across the12 chromosomes.
 Rice genomic DNA was extracted out from each of the landraces of rice using CTAB method.
 DNA samples isolated from each line were quantified on Nano Drop Spectroscopy (NANODROP,
2000c) and the final concentration of DNA was 50 ηg / μl for PCR analysis.
 The volume of the polymerase chain reaction (PCR) was 10µl in Axygen make 96 well PCR plates.
 The profile of the PCR program was as follows: 94°C for 5 min followed by 29 cycles of
94°C for 1 min, 55°C for 1 min, 72°C for1 min with a final extension of 5 minutes at 72°C.
 PCR products were separated in size by 56% polyacrylamide gel electrophoresis and detected by
ethidium bromide.
The size of PCR products were detected by BIORAD gel doc XR + System. The length of each
allele was compared to the standard bands of the standard marker and scored.
46
Genotyping

Molecular characterization :
Genetic associations among 100 accessions were analysed, based on phenotypic
variation of yield traits with the help of 44 SSR markers covering all the
chromosomes.
A total of 217 alleles were amplified and the number of alleles per locus generated
by each marker ranged from 3 to 11 alleles with an average number of 4.93 alleles
per locus.
Maximum number of alleles (11) was amplified by marker RM 164 marker.
 The PIC value across markers ranged from 0.24 to 0.85 with an average of 0.66.
 Maximum PIC value was observed on chromosome 1 (RM164 = 0.85) followed
by RM 248 of chromosome 7 (0.84) and RM 474 of chromosome 1 (0.82).
47

Association analysis between SSR markers and thirteen agronomic and yield attributing traits was
carried out using MLM model over the 100 rice germplasm lines.
 Eleven SSR markers were found to be tightly linked with the panicle length trait. These markers
covered the entire linkage groups except chromosome # 2, 5, 7, 8 and 11.
Five SSR markers namely, RM 133 (C#6), RM 536 (C#11), RM 312 (C#1), RM 474(C#10) and
RM 11 (C#7) were to have tight association with biological yield.
For grain yield two primers, RM 208 (C#2) and RM 247 (C#12) and for harvest index, RM 133
(C#6), RM 208 (C#2) and RM 271 (C#10) were found to be significantly associated with the above
traits.
Three markers viz., RM 474, RM 247 and RM 316 were significantly associated with thousand
grain weight, likewise, six markers showed tightly linked response with number of filled grains.
Similarly, RM 316 and RM 433 for number of unfilled grains and four markers, RM 474, RM 413,
RM 536 and RM 248 showed significant and tight linkage with total number of grains.
48
Results and Discussion

The research provided important information for further mining these elite genes within rice
landraces and using them for rice breeding.
 RM 287 and RM 447 were found to have significant association with flag leaf length and
thousand grain weights, respectively .
In our material, panicle length showed association with RM 283 of chromosome 1.
 In their germplasm accessions, RM 124 also showed significant association with the trait.
The results have clearly shown that structures association mapping in one of the feasible options
to identify major effect QTLs for yield traits in rice.
These marker-trait associations could be further validated and used in marker assisted breeding
for improving particular trait in any rice variety and can be further confirmed in new set of
population as well as in bi-parental mapping population.
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conclusions

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF GENOTYPES DERIVED FROM WILD CROSSES OF PADDY.

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