NAME- K.G.DINUKI ISHARA SIRIWARDANA
REGISTRATION NUMBER-309067913
SUPERVISIORS- PROF. SHYAMA R. WEERAKOON
DR O.V.D.S. JAGATH WEERASENA
DEPARTMENT OF BOTANY ,
THE OPEN UNIVERSITY OF SRI LANKA
Genetic Diversity and the Genetic
Origin of Weedy Rice (Oryza sativa
f. spontanea) growing in rice fields
in districts of Kurunegala and
Matara in Sri Lanka
K.G.D.I.SIRIWARDANA309067913
OUTLINE INTRODUCTION AIMS AND OBJECTIVES METHODOLOGY RESULTS AND DISCUSSION CONCLUSION REFERENCES ACKNOWLEDGMENTS THANK YOU
K.G.D.I.SIRIWARDANA309067913
INTRODUCTION
Rice has been cultivated as a major crop for abouthundreds of years in Sri Lanka
Wild rice
Cultivated rice
Traditional
Inbred
K.G.D.I.SIRIWARDANA309067913
Kaluheenati, kuruwee, gonabaru
Bg, Bw, At, Ld series
Rice ecology in Sri Lanka….
In Sri Lanka, rice is grown under a wide range of physical environments with different elevations, soils and hydrological regimes
The elevation ranges from 0 to 2575m above sea levelTemperature ranges from 300C at sea level to about 150C at the upper most elevations
Rice lands are distributed in almost all agro-ecological environments except for elevations above 2000m
K.G.D.I.SIRIWARDANA309067913
Wild Rice…..
There are 22 wild rice species recorded in worldwide
Five wild rice species were recorded in Sri Lanka
Wild relatives of cultivated rice play an important role in rice breeding
Most diversified crop species due to its adaptation to a wide range of geographical, ecological and climatic regions
K.G.D.I.SIRIWARDANA309067913
Oryza nivara Oryza rufipogon Oryza eichingeri
Oryza granulateOryza rhizomatis
5 wild rice speciesin
Sri Lanka
K.G.D.I.SIRIWARDANA309067913
What is weedy Rice?
Define broadly and genetically,
Conspecific with Oryza sativa that competes cultivated rice and other crops
Weedy rice could occur due to several factors like
• Gene flow between wild and cultivated types• Old rice varieties becoming feral • Crosses between cultivated rice and wild rice
K.G.D.I.SIRIWARDANA309067913
Weedy Rice……
Highly variable and appears to be an intermediate between wild and cultivated rice
Possibly, long-term sympatric distribution has led to similarities between weedy and cultivated rice
Selective herbicides cannot be used to control weedy rice due to its physiological and morphological similarity to the cultivated rice
K.G.D.I.SIRIWARDANA309067913
Specific characteristics
Easy seed shattering and seed dormancy
Taller than the cultivated varieties
Frequently have red pigmented pericarps
Awn present or absent
Vigorous vegetative growth
Comparatively early maturity
High competitive ability
Tolerance to weedicidesK.G.D.I.SIRIWARDANA309067913
Weedy rice are generally taller or as a taller as cultivated rice,Weedy rice has a long awn, Long, thin-grained rice with black hull, Exhibit more rapid seedling growth when compared to the cultivated rice, Internodes color may be purple
Morphological diversity of weedy rice
K.G.D.I.SIRIWARDANA309067913
What problems does weedy rice cause to the rice cultivation
Weedy rice competes with cultivated rice and reducescrop yield
Farmers cannot harvest the grain of weedy rice as ittends to mature earlier and to shatter readily
Therefore, it increases the costs of production, reducesyield, lowers the market value of their rice crop
If not controlled properly can infest crop lands and makethem unfit for rice production
K.G.D.I.SIRIWARDANA309067913
Weedy Rice in Sri Lanka
Weedy rice was reported first in the Eastern province of Sri Lanka in early 1990s, has now spread into many rice growing areas
Weedy rice affects both quantity and quality of national rice production
Therefore, its beneficial to focus a research to assess the level of diversity (agro-morphological and Molecular) of weedy rice population found in districts where weedy rice problem is considerably high
K.G.D.I.SIRIWARDANA309067913
Weedy Rice in Sri Lanka.....
Detailed study of vegetative and reproductive development of weedy rice bio-types can give us the ability to identify weedy rice bio-types at their different growth stages
This will also lead us to know the biology of weedy rice such as life table, growth pattern and life cycle
K.G.D.I.SIRIWARDANA309067913
AIMS AND OBJECTIVES
Aims
Assessment of Genetic Diversity and Genetic Origin of weedy rice (Oryza sativa f.spontanea) growing in rice fields of
Kurunegala and Matara Districts, Sri Lanka
K.G.D.I.SIRIWARDANA309067913
Objectives
Agro-morphological and molecular characterization of weedy rice bio-types and their potential parents in rice fields in Kurunegala and Matara Districts
Tracing the possible genetic origin of weedy rice bio-types in Kurunegala and Matara Districts
Assessment of level of genetic diversity of weedy rice in Kurunegala and Matara Districts
K.G.D.I.SIRIWARDANA309067913
This study was carried out at,
- The Department of Botany, Faculty of Natural Science , Open University of Sri Lanka
- Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo
K.G.D.I.SIRIWARDANA309067913
METHODOLOGY
Materials and Methods
Seeds of weedy rice, Cultivated rice and Wild rice varieties
- Matara- Kurunegala
Two wild rice species from natural habitats - Oryza nivara- Oryza rufipogon
K.G.D.I.SIRIWARDANA309067913
Sample collection
A large number of Weedy rice and cultivated rice
sample collected in Kurunegala and Matara
districts
K.G.D.I.SIRIWARDANA309067913
Sample collection……..
The details of locality of the collecting sites and populations were recorded
The GPS position of each site was recorded with the habitats of the population including the name of the cultivated rice
K.G.D.I.SIRIWARDANA309067913
Table1 – Location and GPS values of Matara District
K.G.D.I.SIRIWARDANA309067913
District Location WR accessions Cultivated
rice varieties
GPS values
Matara Akurugoda MPAW1, MPAW2, MPAW3,
MPAW4
MPAC
(Bg352)
06°03'N,80°33'E
Matara Hakmana MHKW1, MHKW2,
MHKW3, MHKW4, MHKW5
MHKC
(Bg362)
06°08'N,80°65'E
Matara Mapalana MMKW1, MMKW2,
MMKW3, MMKW4
MMKC
(Bg307)
07°15'N,80°01'E
Matara Morawake MMW1, MMW2, MMW3 MMC
(Bg379-2)
06°14'N,08°36'E
Matara Weligama MWMW1, MWMW2,
MWMW3,MWMW4,
MWMW5
MWMC
(Bg379-2)
05°59'N,80°26'E
K.G.D.I.SIRIWARDANA309067913
Table2 – Location and GPS values of Kurunegala District
District Location WR accessions Cultivar
rice
varieties
GPS values
Kurunegala Gokarella KGW1, KGW2 KGC
(Bg352)
07°35'N,80°28'E
Kurunegala Maiwela KMiW1, KMiW2, KMiW3 KMiC
(Bg379-2)
07°38'N,80°30'E
Kurunegala Manapaha KMaW1, KMaW2 KMaC
(Bg352)
07°35'N,80°28'E
Kurunegala Narammala KNW1, KNW2 KNC
(Bg379-2)
07°25'N,80°13'E
Kurunegala Narangalla KNgW1, KNgW2,
KNgW3
KNgC
(Bg352)
07°27'N80°05'E
METHOD
FOR AGRO-MORPHOLOGICAL CHARACTERIZATION
Characterization using the Descriptors forRice
FOR MOLECULAR CHARACTERIZATIONCharacterizing the accession using molecular markers
K.G.D.I.SIRIWARDANA309067913
Identification and labeling of weedy rice accessions and inbred rice varieties
K.G.D.I.SIRIWARDANA309067913
Research design….
Complete Randomized Design (CRD) for Kurunegala District
K.G.D.I.SIRIWARDANA309067913
Agro-morphological Characterization
Data Collection
EXPRESSION OF CHARACTERS
Qualitative: Usually through visual observation e.g. Collar color: 1. absent 2. green 3.light green)
Quantitative: Measurable traits (leaf length, leaf width, etc.)
K.G.D.I.SIRIWARDANA309067913
Data Collection….
Crop Stages
SEEDLING
VEGETATIVE
EARLY REPRODUCTIVE
LATE REPRODUCTIVE
HARVEST
POST HARVEST
K.G.D.I.SIRIWARDANA309067913
Data Collection….
Crop Stages SEEDLING
Seedling Height
VEGETATIVELeaf characteristics (color, pubescence), Ligules, collar, auricles etc
EARLY REPRODUCTIVEFlag-leaf angle (early observation), Culm: Attitude, Node and Inter-node color (early observations, etc.), Spikelet: stigma color, apiculus color, lemma and palea color, awn color (early Observations) etc
K.G.D.I.SIRIWARDANA309067913
Data Collection….
LATE REPRODUCTIVEFlag-leaf angle (late observation), Culm: Node and Inter-node color (late observations), Panicle: type, attitude, secondary branching etc
AT HARVESTSenescence, Panicle Shattering
POST HARVESTPanicle: length, threshability etc, Grain: length, width, pubescence etc
Data were analyzed using SPSS Software package (ver. 16) K.G.D.I.SIRIWARDANA
309067913
Characterization…….
Quantitative
K.G.D.I.SIRIWARDANA309067913
Seed length/width
100 seed weightPanicle length
Filled/unfilled seedsNo of shattered seeds
MOLECULAR CHARACTERIZATION
1) DNA Extraction
2) Quantification and optimization of DNA
concentration
3) Identification and selection of microsatellite/SSR
primers
4) Polymerase chain reaction (PCR)
5) Agarose gel electrophoresis separation and
visualization of PCR products
6) SSR analysis K.G.D.I.SIRIWARDANA309067913
DNA Extraction
Measured 250 mg of leaf material and grind it using mortar and pestle to form a paste
Transferred the ground tissue paste to a new 1.5μl micro-centrifuge tube
Added 400μl of Grinding Buffer
Centrifuged the tube at 14000Χg for 5 minute
Pipette out 150μl of the supernatant into a new 1.5μl micro-centrifuge tube
Added 300μl of Binding Buffer
Added 130μl of Precipitation Buffer incubate on ice for 5 minutes
K.G.D.I.SIRIWARDANA309067913
DNA Extraction…..
Centrifuged the tube at maximum speed for 10 minutes
Transferred supernatant to sterile 1.5ml centrifuge tube
Added 1.5 volumes Binding Buffer
Applied 650μl of mixture to a Phytospin column and centrifuged for 1-10 minutes
Added 500μl of Wash Buffer 1 to the column and centrifuged the tube at 5000Χg for 2 minutes
Discarded the flow-through liquid from the collection tube
Added 500μl of Wash Buffer 2 to the column and Centrifuged the tube at 5000Χg for 1 minutes
K.G.D.I.SIRIWARDANA309067913
DNA Extraction…..
Discarded the flow-through liquid from the collection tube
Added 500μl of Wash Buffer 2 to the column and Centrifuged the tube at 5000Χg for 1 minutes
Discarded the flow-through liquid from the collection tube
Placed the empty collection tube and the column in the centrifuge
Centrifuged at 12000Χg for 1 minute
Placed the Phytospin column in a new micro-centrifuge tube
Added 50μl of Elution Buffer K.G.D.I.SIRIWARDANA309067913
DNA Extraction…..
Incubated at room temperature for 1 minute
Centrifuged the tube at 5000Χg for 1 minute to elute the DNA
Plant DNA can be used immediately for any downstream application or stored at 20ºC until use
K.G.D.I.SIRIWARDANA309067913
Quantification and optimization of DNA concentration
The amount of genomic DNA was quantified at
260nm spectrophotometrically
Using the absorbance reading was obtained for
DNA sample of each rice variety, the
original DNA concentrations were
determined and adjusted to 50ng/μl
K.G.D.I.SIRIWARDANA309067913
Identification and selection of microsatellite/SSR primers Eight SSR primer pairs designed from cultivated rice were
identified to assay genetic variation of all included
materials, based on the Rice Genes Database
Out of eight SSR Primer Pairs, two microsatellite primer pairs
(RM21 and RM11) were selected as a subset of SSR
markers previously used to assay genetic diversity of wild
rice and WR by specific polymerase chain reaction (PCR)
conditions
The selected Primers were screened against selected weedy,
cultivar and wild varieties at a time K.G.D.I.SIRIWARDANA309067913
Identification and selection of microsatellite/SSR primers…..
Table 3 - SSR primer pairs used for DNA amplification
The primer was labelled at its 5'-end with one of the following fluorescent dyes: VIC, FAM, NED and PET (Applied Biosystems)
Table 4 - Fluorescent dyes
K.G.D.I.SIRIWARDANA309067913
Polymerase chain reaction (PCR)
The polymerase chain reactions were performed in a PTC
10096v thermocycler programme
A denaturation period of 4 min at 94 ⁰C was followed by 36
cycles of 30 s at 94 ⁰C, 30 s at 55 ⁰C and 1 min. at 72 ⁰C,
and then 10 min at 72⁰C for the final extension
Reactions were carried out in a volume of 25µL containing
1X buffer, 1.5mM each of dNTPs, 10mM of SSR primer,
50 ng of genomic DNA and 0.5U of Taq polymerase
K.G.D.I.SIRIWARDANA309067913
Polymerase chain reaction (PCR)…..
PCR Reagents• 1X Buffer
– 10mM Tris-HCl, 50mM KCl
• MgCl2– 1mM - 4mM (1.5mM)
• dNTPs– 1.5mM
• Primers– 10μM
• DNA polymerase – Taq DNA polymerase is thermostable– 1-4 Units (1 unit)
• DNA– 10pg-1μg (50ng)
Primers
DNA template
Buffer
+ +
A C T G
MgCl2
Taq polymerasedNTPs
Polymerase chain reaction (PCR)…..
Some steps involved in PCR
K.G.D.I.SIRIWARDANA309067913
This whole process was done by using an automated machine called as thermo cycler
PCR tube
Agarose gel electrophoresis separation and visualization of PCR products
Agarose Buffer Solution
Melting the Agarose
Combine the agarosepowder and buffer solution
Agarose is insoluble at room temperature (left)The agarose solution is boiled until clear (right)
visualization of PCR products…..
Some steps involved in visualization of PCR products
K.G.D.I.SIRIWARDANA309067913
Agarose gel electrophoresis…..
Overview of Agarose gel electrophoresis separation and visualization of PCR products
K.G.D.I.SIRIWARDANA309067913
SSR analysis
The DS – 33 Matrix standards is used to
generate the multi-component matrix
required with analyzing 6 – FAM, VIC,
NED, PET and Gs600 LIZ dye label DNA
fragments on the applied Bio-system 3500
The data collection software for these
instruments uses the multi-component
matrix to automatically analyze the 5
different color florescent dyes – label
samples in a single capillaryK.G.D.I.SIRIWARDANA309067913
SSR analysis…..
K.G.D.I.SIRIWARDANA309067913
RESULTS AND DISCUSSION
FOR AGRO-MORPHOLOGICAL CHARACTERIZATION
FOR MOLECULAR CHARACTERIZATION
K.G.D.I.SIRIWARDANA309067913
Agro-morphological characters
Classification and Regression Tree Analysis (CART)
K.G.D.I.SIRIWARDANA309067913
Agro-morphological characters
Dendrogram of hierarchical clustering using dissimilarity values from agro-morphological characteristics of WR, cultivated rice and wild rice
K.G.D.I.SIRIWARDANA309067913
Method of analysis Character selection
CART analyses Seed coat color at maturity, Leaf senescence,
Panical threshability, Culm number after full heading,
Culm length (cm), Number of leaves,
Leaf blade pubescence at late vegetative stage,
Awning after full heading, Panicle length (cm)
Cluster
analyses
Panical axis at maturity, Sterile lemma color,
Internode color after flowering,
Secondary branching, Panicle length (cm),
Leaf blade width (cm), Culm number after full heading,
Culm strength (lodging resistance),
Awning after full heading, Number of leaves,
Panical exsertion, Awn length (cm),
Leaf senescence,
100 Grain weight at maturity (g),
Lemma and palea color at maturity,
Culm angle after flowering
Matara District
Dendrogram of hierarchical clustering using dissimilarity values from agro-morphological characteristics of observed WR, Wild rice and rice cultivars in MataraDistrict
K.G.D.I.SIRIWARDANA309067913
Kurunegala District
Dendrogram of hierarchical clustering using dissimilarity values from morphological characteristics of observed WR, Wild rice and rice cultivars in KurunegalaDistrict
K.G.D.I.SIRIWARDANA309067913
Both Kurunegala District and Matara Districts
Dendrogram of hierarchical clustering using dissimilarity values from agro-morphological characteristics of observed WR, Wild and rice cultivars in Matara and KurunegalaDistricts
K.G.D.I.SIRIWARDANA309067913
Visualization of PCR products
Matara District
PCR amplification products from, 7 weedy rice, 5 cultivated rice and 2 wild rice genomic DNA with primer pair RM21. Lane 1 – MPAW2, Lane 2 – MMW2, Lane 3 –MHKW3, Lane 4 – MMKW3, Lane 5 – MPAW1, Lane 6 – MMKW2, Lane 7 - MMW1, Lane 8 - MMC, Lane 9 –MMKC, Lane 10 – MHKC, Lane 11 –MWMWC, Lane 12 –MPAC, Lane 13 – O.nivara, Lane 14 – O.rufipogon
PCR amplification products from, 7 weedy rice, 5 cultivated rice and 2 wild rice genomic DNA with primer pair RM11. Lane 1 – MPAW2, Lane 2 – MMW2, Lane 3 –MHKW3, Lane 4 – MMKW3, Lane 5 – MPAW1, Lane 6 – MMKW2, Lane 7 -MMW1, Lane 8 - MMC, Lane 9 –MMKC, Lane 10 – MHKC, Lane 11 –MWMWC, Lane 12 – MPAC, Lane 13 – O.nivara, Lane 14 – O.rufipogon K.G.D.I.SIRIWARDANA
309067913
Kurunegala District
PCR amplification products from, 5 weedy rice, 3 cultivated rice and 2 wild rice genomic DNA with primer pair RM21. Lane 1 – KMaW1, Lane 2 – KGW2, Lane 3 – KNW1, Lane 4 –KNgW1, Lane 5 –KMiW1, Lane 6 – KNC, Lane 7 - KMaC, Lane 8 –KGC, Lane 9 –O.nivara, Lane 10 – O.rufipogon
PCR amplification products from, 5 weedy rice, 3 cultivated rice and 2 wild rice genomic DNA with primer pair RM11. Lane 1 – KMaW1, Lane 2 – KGW2, Lane 3 – KNW1, Lane 4 –KNgW1, Lane 5 –KMiW1, Lane 6 – KNC, Lane 7 - KMaC, Lane 8 –KGC, Lane 9 –O.nivara, Lane 10 – O.rufipogon
K.G.D.I.SIRIWARDANA309067913
SSR genotyping score
Chromatogram of the SSR genotyping. Amplicons with high content of DNA and reduced quantities of unspecific amplifications, shutter bands and noise K.G.D.I.SIRIWARDANA
309067913
GENETIC DIVERSITY AND GENETIC ORIGIN OF WEEDY RICE (Oryza sativa f. spontanea)
Relatively high genetic diversity is found in
Matara and Kurunegale weedy rice populations
Considerable genetic differentiation is present among
weedy rice populations in Matara and
Kurunegale Districts
The possible origin of weedy rice in Matara and
Kurunegale Districts based on its genetic
relationships K.G.D.I.SIRIWARDANA309067913
CONCLUSION Out of forty seven agro-morphological characters twenty
two characters have identified as top key characters
Different bio-types are identified in Matara (six bio-types)
and Kurunegala (three bio-types) Districts
Higher diversity among WR accessions indicates that there
would be a higher possibility of out-crossing rate
between cultivated rice varieties and their weedy
relatives which could lead to establish WR as a
prominent weed
K.G.D.I.SIRIWARDANA309067913
CONCLUSION…..
Molecular characterization of present study with
selected bio-types confirmed the results
obtained from agro-morphological characters
Agro-morphological and molecular studies are
necessary to cover all over the country to get a
better picture of diversity and origin of weedy
rice
K.G.D.I.SIRIWARDANA309067913
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ACKNOWLEDGEMENTSI acknowledge the guidance given by Prof. Shyama R. Weerakoon, Dr O.V.D.S. Jagath
Weerasena and Dr S. Somaratne in carrying out this research project and providing facilities to
conduct this research at the Department of Botany, Faculty of Natural Science, Open
University of Sri Lanka and Institute of Biochemistry, Molecular Biology and
Biotechnology (IBMBB) University of Colombo
K.G.D.I.SIRIWARDANA309067913