genetic diversity and the genetic origin of weedy rice (oryza sativa f. spontanea) growing in rice...

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


INTRODUCTION

Rice has been cultivated as a major crop for abouthundreds of years in Sri Lanka

Wild rice

Cultivated rice

Traditional

Inbred


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


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


Oryza nivara Oryza rufipogon Oryza eichingeri

Oryza granulateOryza rhizomatis

5 wild rice speciesin

Sri Lanka


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


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


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


Early shattering of

grains

Handgrip test for

shattering of weedy rice


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


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


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


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


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


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


METHODOLOGY

Study Sites


Kurunegala

Matara

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


Sample collection

A large number of Weedy rice and cultivated rice

sample collected in Kurunegala and Matara

districts


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


Table1 – Location and GPS values of Matara District


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


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


Identification and labeling of weedy rice accessions and inbred rice varieties


Germination of seeds


Young seedlings


Planted in the portable trays to grow the seedlings for 25 days


Seedlings were transplanted to plastic pots


Research design

Complete Randomized Design (CRD) for Matara District


Research design….

Complete Randomized Design (CRD) for Kurunegala District


Plant house experiment conducted at OUSL, Nawala


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.)


Data Collection….

Crop Stages

SEEDLING

VEGETATIVE

EARLY REPRODUCTIVE

LATE REPRODUCTIVE

HARVEST

POST HARVEST


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


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…….


Qualitative


Quantitative


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


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


DNA Extraction…..


Added 500μl of Wash Buffer 2 to the column and Centrifuged the tube at 5000Χg for 1 minutes


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


DNA Extraction…..

Some steps involved in DNA Extraction


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


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


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


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

DNA amplification

using PCR


Polymerase chain reaction (PCR)…..

Some steps involved in PCR


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)

Agarose gel electrophoresis…..


visualization of PCR products…..

Some steps involved in visualization of PCR products


Agarose gel electrophoresis…..

Overview of Agarose gel electrophoresis separation and visualization of PCR products


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…..


http://www.google.lk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=D-0rtnMI2gCMUM&tbnid=mBrdJ8KdgHgG2M:&ved=0CAUQjRw&url=http://www.biocenter.helsinki.fi/bi/dnagen/abi3130.htm&ei=Jp_fUo31JsfprQe7iYCoDg&bvm=bv.59568121,d.bmk&psig=AFQjCNEkXITxrA8JMO6ISq83FL8WlbDTjw&ust=1390473312438637

http://www.google.lk/url?sa=i&rct=j&q=&esrc=s&frm=1&source=images&cd=&cad=rja&docid=D-0rtnMI2gCMUM&tbnid=mBrdJ8KdgHgG2M:&ved=0CAUQjRw&url=http://www.biocenter.helsinki.fi/bi/dnagen/abi3130.htm&ei=Jp_fUo31JsfprQe7iYCoDg&bvm=bv.59568121,d.bmk&psig=AFQjCNEkXITxrA8JMO6ISq83FL8WlbDTjw&ust=1390473312438637

RESULTS AND DISCUSSION

FOR AGRO-MORPHOLOGICAL CHARACTERIZATION

FOR MOLECULAR CHARACTERIZATION


Agro-morphological characters

Classification and Regression Tree Analysis (CART)


Summary of CART analysis

Agro-morphological characters

Dendrogram of hierarchical clustering using dissimilarity values from agro-morphological characteristics of WR, cultivated rice and wild rice


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


Kurunegala District

Dendrogram of hierarchical clustering using dissimilarity values from morphological characteristics of observed WR, Wild rice and rice cultivars in KurunegalaDistrict


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


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


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


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


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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


THANK YOU


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