Genotyping of doubled haploid population using SSR markers

for QTLs mapping

Submitted To: Submitted By:

Dr. Navraj Kaur Sarao Anroop kaur

Assistant Plant Breeder (L-2016-A-5-BIOTECH LE )

School of Agriculture Biotechnology,

PAU, Ludhiana

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 CERTIFICATE

This is to certify that the project, “Genotyping of Double Haploid population using
SSRs markers for QTLs mapping”, carried out as In house project in the course
Biotech.499 has been undertaken by Anroop kaur in my supervision.

Dr. Johar Singh Saini

Advisor

Director
School of Agricultural Biotechnology
PAU, Ludhiana

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 Acknowledgement

With profound gratitude and immense pleasure we would like to express our sincere thanks to our project advisor
Dr. Johar Singh for his valuable advice and supervision. His constant encouragement and confidence in us to take
on the various responsibilities has always been a reason for us to work harder. We would like to expand our
thanks to Sorabh Sethi and Puneet kaur for their guidance throughout the project

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INDEX

TOPICS PAGE NO.

Introduction 5

Objectives 6
Experiments 6
 DNA Extraction 6-9
 DNA quantification 9-10
 PCR analysis 11

 Primers 12
 Gel electrophoresis 13-14
 Screening of polymorphic SSRs 14
 Genotyping of Double haploid population 14
 Screening of gamma mutant plant 15
 Future aspects 16
 References 17-18

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TITLE: Genotyping of doubled haploid population using SSR markers for QTLs
mapping

INTRODUCTION:

Wheat ( Triticum aestivum L.) is the widely grown cereal in temperate and some tropical
environments, where is often cultivated as the winter season crop in rotation with number of
other crops. As with most crops, wheat yields are also vulnerable to climatic stresses like high
temperature stress, cold stress and water stress. Under the changing climate Increase in
temperature particularly during reproductive phase could negatively affect global wheat yields
threating the food security. The most important reproductive trats effected by high temperature
are floral fertility, grain number and weight, and overall grain yield. High temperature also
effects physiological responses including premature leaf senescence, reduced photosynthetic
rate, reduced seed set. Genetic variation is available for the component traits, however,
underlying genetic factors and precise locations are need to mapped for easy handling of this
complex traits. Review of the literature on genetics of heat stress in wheat showed that there are
only few QTLs are mapped explaining, low phenotypic variation. Usually the markers flanking
the QTLs are present in away from the structural part of the gene that control phenotype, mostly
PCR based and SSRs showing loose linkage, low in number and laboriousto apply. Therefore,
there is need to develop markers which are easy to apply, abundant in number and preferably
from genic regions. Developing SNPs using genotyping by sequencing, which is high
throughout, simple and cost effective. However, use of the SSRs will help in anchoring the
regions with SNPs linked to the QTLs. Therefore, genotyping SSRs will help in finding the
QTLs for various reproductive traits on one hand and anchoring the regions with SNPs linked to
these QTLs. This technique is useful for a crop like wheat with a large genome with genes
present in localized gene-rich regions.
Therefore, the main goal of the project was find the polymorphic SSRs and their applications on
the doubled haploid wheat population to identify the QTLs responsible for providing tolerance to
heat stress.

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 OBJECTIVES:

 To find polymorphic SSRs between two parental lines PBW343 and

SUNSTAR

 Genotyping of doubled haploid mapping population with polymorphic SSRs

EXPERIMENTS:

 Experiment 1: Isolation of DNA of parental lines and doubled haploid population.

 Experiment 2: polymorphic survey between parent lines using SSRs
 Experiment 3: Genotyping of Doubled haploid population.

DNA Extraction-

Isolation of DNA is a prerequisite for all DNA manipulations. In plants, DNA can be
isolated from any plant part. However, young tissues are the best source for good quality
DNA. All plant DNA extraction protocols comprise of the basic steps of disruption of
the cell wall, cell membrane and nuclear membrane to release the DNA into solution
followed by precipitation of DNA while ensuring removal of the contaminating
biomolecules such as the proteins, polysaccharides, lipids, phenols and other secondary
metabolites. This is brought about by disruption of the tissue in a mortar and pestle aided
by liquid nitrogen and the various components of the homogenization or extraction buffer
followed the precipitating and purification methods employed. Since DNA can be
extracted from various types of tissues such as seedlings, leaves, cotyledons, seeds,

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endosperm, tissue culture callus, roots etc., the tissue type along with the concentration of
DNA finally required determine the methodology of DNA extraction to be followed.

DNA of the parental lines and DH plants will be isolated from young leaves following
SDS method.

DNA EXTRACTION BUFFER:

Final conc. Vol/liter

1M Tris, pH 8.0 100mM 100ml
5M Nacl 500mM 100ml
0.5M EDTA 50mM 100ml
20% SDS 0.84%(w/v) 62.5%

Make the volume to 1 L with Double Distilled Water.

Purpose of Extraction:

To obtain DNA in a relatively purified form which can be used for further investigations,
i.e. PCR, sequencing, etc

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 Procedure

 Grind fresh leaves (any tissue can be used) to a fine powder. Use liquid if grinding
with mortar and pistil, Transfer the fine powder up to about 15-20 ml mark in a 50
ml polypropylene tube.
 Add sodium bisulfate to extraction buffer and adjust pH to 7.8-8.0. Heat the
extraction buffer to 65C and add about 20-25ml to each tube. Mix with a spatula
breaking all the clotes to make a fine slurry. Incubate in a water bath at 65°C for
30 – 60 minutes, inverting tubes every 5-10 min.
 Add chloroform : isoamyl alcohol (24:1) to about 40 ml of the tubes , cap them and
mix gently but thoroughly to produce an emulsion.
 Centrifuge at room temperature at 5-8 K for 15 minutes. Pour upper phase into a
clean 50ml tube.
 Add 2 volumes absolute ethanol. Mix gently to get a DNA clot .
 Remove the solution by retaining the DNA clot in the tube. Wash DNA with 20ml
of 70% ethanol. Leave at room temperature for 20 minutes, mix occasionally,
Repeat the wash if needed.
 Drian ethanol, air dry the DNA pellet, and add 1ml TE+ 1ul of RNAase
(10mg/ml). Leave at 4C overnight. Next day, incubate the tubes in 65C water bath
until DNA is dissolved. Occasional mixing and/or vortexing may be needed to
completely dissolve DNA. It may take several hours.
 When DNA is completely dissolved add 1ml of phenol:chloroform (1:1) into the
tube. Mix thoroughly but gently and transfer the mixture to 2ml eppendrof tube.
Spin at room temperature in a microcentrifuge for 5 minutes.
 Transfer the supernatant to new tube using a wide bore pipet tip.
 Add 1/10th volume of 3M NaAc, pH 5.5 and 1 volume of propanol mix well.

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  Drain the solution by retaining the pellet in tube . Fill the tube with 70% ethanol
and leave for at least 30min. The pellet may also be left in 70% ethanol overnight
at 4C
 Drian the ethanol and dry the pellet at room temperature for about an hour.
Resuspend the pellet in an amount of TE dependent upon the size of the DNA
pellet.
 Determine the DNA concentration by running a 1:40 dilution{DNA :(3TE:1
loading buffer)} on an agarose gel along with a conc. Marker (uncut lambda DNA
at conc. Of 5,10,20 and 50ng). Stain with 10ul ethdium bromide for 15 minutes
and photograph. Visually compare the intensity of the DNA bands with markers to
determine the concentration

.DNA Quantification

DNA quantification can be done through Spectrophotometry or Agarose gel

electrophoresis method. DNA was quantified by gel visualization method. This involved
running DNA samples on 2.5% agarose gel (15gm agarose in 600ml of 50X TBE
buffer),ethidium bromide (0.5μg/mL) was added for visualization of the samples under
UV light. 2ul of DNA and 8ul of bromophenol dye was added in a fresh/autoclaved plate.
Samples were then loaded on 2.5% gel, run at 120V for 2 hrs. The gel was viewed in gel
documentation system. Intensities were compared and nucleic acid concentrations were
estimated.

Genomic DNA was diluted to a concentration of 25ng/µL using autoclaved distilled

water. Dilutions were made by using the formula:

n1v1 = n2v2

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where,n1 is the concentration of sample, v1 is the initial volume, n2 is the final cond v2 is
the final volume.

DNA quantification

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 PCR analysis

PCR is a rapid procedure for in-vitro enzymatic amplification of a specific segment

of DNA using specific primer sequences. The reaction mixture consists of three nucleic
acid segments: target DNA (double stranded) and two oligonucleotide primers (single
stranded). Additionally, there is a protein component (DNA polymerase enzyme),
deoxyribonucleoside triphosphate (dNTPs), a buffer and salts.

PCR Profile:

Steps Temperature Time

1. Initial Denaturation 94⁰C 4 min
2. Denaturation 94⁰C 1 min
Repeat step (2-4)
3. Annealing 55⁰C 1 min
34 times
4. Extension 72⁰C 1 min
5. Final Extension 72⁰C 7 min
6. Hold 4⁰C ∞

Reaction mixture:

S.NO. COMPONENTS Stock sol.(ul) Working sol.

1 DNA 2 2
2 Buffer 2 200
3 MgCl 1 100
4 dNTPs 0.25 25
5 Primer (F+R) 2 200
6 Taq 0.2 20
7 Water 2.55 255

Final reaction volume=10ul

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Primers

SSR markers also known as microsatellites are smallest in size (2-8bp repeats) and are
highly polymorphic codominant markers. They show polymorphism even within species
as the number of repeats varies from individual to individual. They have been
successfully used as DNA markers to estimate genetic diversity, genome mapping and
marker assisted selection for agronomically important traits.

List used SSR markers:

Barc0183 wmc0052
wmc0027 barc0049
Barc0219 barc0267
Gwm0484 wmc0819
Cfd0141 barc1090
wmc0491 wmc0790
Barc0080 Barc0320
Gwm0099 Wmc0819

Gwm0058

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 Gel Electrophoresis

Principle

It is a method used to separate a mixture of DNA or protein on the matrix of agarose. The
protein molecules are separated by charge and\or size, the DNA and RNA fragments by
length. The biomolecules are moved by applying an electric field to move the charged
particles on the matrix of agarose. The biomolecules are separated by size on the matrix
of agarose.

𝑬 ∝ 𝟏/µ

E=electric field applied(V/cm)

µ=size of charged particle

Procedure:

 3µl of loading dye was added in each PCR product

 Both edges of a gel mold were sealed with masking tape. Placed in level platform
and combs attached.
 Prepared 3% agarose by dissolving 15g agarose in 500ml of 0.5X TBE buffer.
 Boiled for 6 min in a microwave. Allowed the solution to cool to 60⁰C
 Added ethidium bromide at a concentration of 0.5µg/ml.
 Poured agarose into gel mold and allowed to solidify.
 Filled the electrode tank with 0.5x TBE buffer.
 Removed masking tape from both the ends of the gel mold. Mounted the gel mold
on to the electrode tank making sure no bubbles are formed beneath the mold.
 The combs were gently removed.
 Loaded 3µl of 50bp DNA ladder in the first well and 10µl of each reaction mixture
in the succeeding wells.

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 Closed the tank and attached electrode wires to the power supply. Run for 3-4 hr at
150V.
 View gel in a gel documentation system

Screening of polymorphic SSRs

 Number of SSRs have screened for genetic polymorphism using panel of wheat
varieties.

• Available a set of genome marker is used.

• Primers were first screened on the parental lines, those which were polymorphic
then screened on the bulks.

Genotyping of double haploid population

 Scoring of DNA is done with the help of 16 primers on 180 samples.

 DNA was diluted to 25ng/µl to prepare the dilution plate for PCR.
 Here, we used 2.5% agarose to score the DNA with parents PBW343× SUNSTAR

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 DOUBLE HAPLOID POPULATION

This DH population has 200 entries including 2 parents ie PBW343×SUNSTAR,

Also this population used for DNA extraction and screening of gamma mutants.

Screening of Gamma mutation

• Mutation- changes in genes and chromosomes.

• Have bulk seed of M1 plant harvested in previous year.

• And use 4 times the no. of seed of M1 generation.

• To find out mutant plants, gave Gamma radiations with lower energy levels are
capable of causing excitation at the level of nitrogen bases of the level of the
genetic material.

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 Example of mutant plants

FUTURE ASPECT

• Genotyping and polymorphoric markers will help in genotyping linkage and will
be a part of QTL mapping project.

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