Practical handouts for The Regional Training Course on Utilization of Molecular Markers for PGRFA Characterization and Pre-Breeding for Climate Changes

Practical Handouts

for

The Regional Training Course

on

Utilization of Molecular Markers for PGRFA

Characterization and Pre-Breeding for

Climate Changes

Agricultural Genetic Engineering Research

Institute

EGYPT

(August 31 to Sept. 4, 2014)

Utilization of Molecular Markers for PGRFA Characterization and Pre-Breeding for Climate Changes

AGERI, Aug. 31-Sept. 4, 2014 Page 2

Genomic DNA Extraction from Plant Tissues

“CTAB” Method

Deoxyribonucleic acid (DNA) isolation is an extraction process of DNA from various

sources. Methods used to isolate DNA are dependent on the source, age, and size of the sample.

Despite the wide variety of methods used, there are some similarities among them. In general,

they aim to separate DNA present in the nucleus of the cell from other cellular components.

The isolation of DNA usually begins with lysis, or breakdown, of tissue or cells. This

process is essential for the destruction of protein structures and allows for release of nucleic

acids from the nucleus. Lysis is carried out in a salt solution, containing detergents to denature

proteins or proteases (enzymes digesting proteins), such as Proteinase K, or in some cases both.

It results in the breakdown of cells and dissolving of membranes.

Presence of proteins, lipids, polysaccharides and some other organic or inorganic

compounds in the DNA preparation can interfere with DNA analysis methods, especially with

polymerase chain reaction (PCR). They can also reduce the quality of DNA leading to its shorter

storage life.

Plants are more difficult to work with than many other materials for a couple of reasons.

First, plant cells have a cell wall, which has to be at least partly destroyed before the cytoplasm

with the DNA can be accessed. Second, plants often have high levels of sugars (for example

starch or fructose) in their tissues or other organic compounds such as polyphenols.

Grinding of the samples in liquid nitrogen helps to destroy the cell wall, but the organic

compounds including sugars still remain. As a result, methods were developed that use

chloroform-isopropyl alcohol mix, (CTAB) hexadecyltrimethylammonium bromide with high

salt to remove polysaccharides, and polyvinylpyrrolidone (PVP) to remove polyphenols.

In order to purify DNA, insoluble particulates are removed through centrifugation while

soluble proteins and other material are separated through mixing with chloroform and

centrifugation. DNA must then be precipitated from the aqueous phase and washed thoroughly to

remove contaminating salts. The purified DNA is then resuspended and stored in TE buffer or

sterile distilled water. This method has been shown to give intact genomic DNA from plant

tissue. To check the quality of the extracted DNA, a sample is run on an agarose gel, stained with

ethidium bromide, and visualized under UV light.



Procedure

With this method, from 50 to 100 ug of DNA per each 100 mg leaf tissue may be obtained.

When extracting DNA from larger amounts of tissue, increase the amounts given below (up to

1000 mg).

1. Place 100mg of lyophilized ground leaf tissue in a 2.0 ml tube (if using a 1.5 ml tube, all

volumes may be scaled down by 25%).

2. Add 1 ml of CTAB isolation buffer. Mix by gentle swirling to homogenize the tissue with

the buffer.

3. Incubate the samples at 65°C for 30 min with continuous gentle rocking or mix gently per

10 min.

4. Centrifuge at 10,000 rpm at room temperature for 10 min to generate an aqueous phase

and an organic phase.

5. Remove approximately 750 µl of the aqueous phase and place in a new 1.5 or 2.0 ml tube

containing 5 µl RNAse.

6. Mix with gentle inversion and incubate at 37°C for 30 min.

7. Add 500 µl of chloroform: isoamyl (24:1). Mix gently with continuous rocking for 10

min at room temperature.

8. Centrifuge at 10,000 rpm for 10 min.

9. Transfer upper phase to new new 1.5 tube.

Optional step: Repeat the chloroform treatment on the aqueous phase. This produces

cleaner DNA, but a lower yield.

10. Add equal volume ice-cold 100% isopropanol (2-propanol). Mix very gently to

precipitate the nucleic acid.

Optional step: Incubate samples at -20°C overnight, especially if precipitated DNA is not

visible.

11. Centrifuge at 10,000 rpm for 10 min.

12. Add 500 µl of 75% ethanol. Wash the DNA pellet gently.

13. Centrifuge at 10,000 rpm for 2-3 min.

14. Pour off liquid (being careful not to lose pellet).

Optional step: Wash once again for further purification.

15. Allow pellet to air-dry until ethanol evaporates completely. Any remaining alcohol smell

indicates pellet is not completely dry.

16. Re-suspend the DNA pellet in 50-100 µl of TE or double-distilled water. Store samples at

4°C until use; if DNA will not be used for a long time, store at -20°C.



Stock Solutions

CTAB extraction buffer

0.5 M EDTA-8.0

Dissolve 186.12 g Na2EDTA•2H20 (MW=372.24) in approx. 750 ml of dH2O. Add NaOH

pellets to bring pH to 8.0. After EDTA is in solution, bring to 1000 ml with dH2O.

Autoclave.

5 M NaCl: 5 M sodium chloride

Dissolve 292.2 g NaCl (MW=58.44) in dH2O to a final volume of 1000 ml. Autoclave.

1 M Tris - pH 7.5, 8.0 or 9.5 Dissolve 121 g Tris-Base in approx. 750 ml dH2O. Add conc. HCl until desired pH is

reached (75 ml HCl = pH 7.5, 49 ml HCl = pH 8.0). Bring solution to 1000 ml with dH2O.

Autoclave.



PCR Optimization

Reaction Conditions and Components

The GeneAmp PCR process is widely employed in a tremendous variety of

experimental applications to produce high yields of specific DNA target sequences.

Sample Volume and Reaction Tubes:

Most PCR protocols are performed at the 25 μL - 50 μLscale in 0.2 mL or 0.5 mL

microcentrifuge tubes.

PCR Conditions:

Component Component volume Final concentration

Template DNA variable ˂0.5µg/50 µl

Upstream primers 5- 50 pmol 0.1-1.0 µM

Downstream primer 5- 50 pmol 0.1-1.0 µM

PCR nucleotide Mix (dNTPs),

10 mM each

1 µl 200µM each

Taq DNA polymerase (5u/ µl) 0.25µl 1.25u/50 µl

PCR Buffer 10 µl 1x

MgCl2 25 mM solution 2.0- 8.0 µl 1.0- 4.0 mM

Nuclease free water to finalize the volumeto 50 µl



Analysis:

PCR products are usually analyzed by staining with ethidium bromide and transillumination

under short- wave UV light.



Amplified Fragment Length Polymorphism (AFLP)

A technique for DNA fingerprinting

The AFLP technique is based on the selective PCR amplification of restriction fragments from a

total digest of genomic DNA. The technique involves three steps:

(i) Restriction of the DNA and ligation of oligo-nucleotide adapters,

(ii) Selective amplification of sets of restriction fragments, and

(iii) Gel analysis of the amplified fragments. PCR amplification of restriction fragments is

achieved by using the adapter and restriction site sequence as target sites for primer

annealing.

The selective amplification is achieved by the use of primers that extend into the restriction

fragments, amplifying only those fragments in which the primer extensions match the

nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be

visualized by PCR without knowledge of nucleotide sequence. The method allows the specific

co-amplification of high numbers of restriction fragments. The number of fragments that can be

analyzed simultaneously, however, is dependent on the resolution of the detection system.

Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide

gels.

The AFLP technique provides a novel and very powerful DNAfingerprinting technique for

DNAs of any origin or complexity.

Reference:

Pieter Vos et al,1995, Vol. 23, No. 21 4407-4414.





Summary of the AFLP® Analysis System I

The AFLP® Analysis System I has been designed for use with plants having genomes ranging in

size from 5 × 108 to 6 ×109 bp, including tomato, corn, soybean, cucumber, lettuce, barley,

cotton, oilseed rape, potato, sunflower, pepper, and brassica. The AFLP® technique involves

three major steps:

1. Restriction endonuclease digestion of the DNA and ligation of adapters. 2. Amplification

of the restriction fragments.

3. Gel analysis of the amplified fragments.

Typically, 50 to 100 restriction fragments are coamplified in each AFLP® reaction and detected

by denaturing gel electrophoresis. This technique is, therefore, extremely powerful for the

identification of DNA polymorphisms.

Overview: (Invitrogen,life technologies Instruction Manual, AFLP® Analysis

System I Kit)

1. Restriction Endonuclease Digestion

To prepare an AFLP® template, genomic DNA is isolated and digested with two restriction

endonucleases simultaneously. This step generates the required substrate for ligation and

subsequent amplification.

The restriction fragments for amplification are generated by two restriction endonucleases: EcoR

I and Mse I. EcoR I has a 6-bp recognition site, and Mse I has a 4-bp recognition site. When used

together, these enzymes generate small DNA fragments that will amplify well and are in the

optimal size range (<1 kb) for separation on denaturing polyacrylamide gels. Due to primer

design and amplification strategy, these EcoR I–Mse I fragments are preferentially amplified

(rather than EcoR I–EcoR I or Mse I–Mse I fragments).

The success of the AFLP® technique is dependent upon complete restriction digestion; therefore,

much care should be taken to isolate high quality genomic DNA, intact without contaminating

nucleases or inhibitors.

2. Ligation of Adapters

Following heat inactivation of the restriction endonucleases, the genomic DNA fragments are

ligated to EcoR I and Mse I adapters to generate template DNA for amplification. These



common adapter sequences flanking variable genomic DNA sequences serve as primer binding

sites on these restriction fragments. Using this strategy, it is possible to amplify many DNA

fragments without having prior sequence knowledge.

3. Amplification Reactions

PCR is performed in two consecutive reactions. In the first reaction, called preamplification,

genomic DNAs are amplified with AFLP® primers each having one selective nucleotide. The

PCR products of the preamplification reaction are diluted and used as a template for the selective

amplification using two AFLP® primers, each containing three selective nucleotides. (The EcoR

I selective primer is 32P- or 33P-labeled before amplification.) This two-step amplification

strategy results in

consistently cleaner and more reproducible fingerprints with the added benefit of generating

enough template DNA for thousands of AFLP® reactions.

The most important factor in determining the number of restriction fragments amplified in a

single AFLP® reaction is the number of selective nucleotides in the selective primers. The

selective primers in the AFLP® Analysis System I contain three selective nucleotides. In

practice, using the AFLP® Analysis System I with plants having genomes ranging in size from

5.108 to 6.10

9 bp, the number of fragments amplified per sample per primer pair averages 50, but

may range from as low as 10 to ~100 depending on the sequence context of the selective

nucleotides, and the complexity of the genome (1,2,10).

A second factor in determining the number of amplified fragments is the C and G composition of

the selective nucleotides. In general, the more Cs and Gs used as selective nucleotides in the

amplification primers, the fewer DNA fragments amplified.

Also, the smaller the genome being analyzed, the fewer fragments amplified and the simpler the

fingerprint.

4. Separation of Amplified Fragments on Denaturing Polyacrylamide Gels

Products from the selective amplification are separated on a 5% or 6% denaturing

polyacrylamide (sequencing) gel. The resultant banding pattern (“fingerprint”) can be analyzed

for polymorphisms either manually or using analytical software (1,2).



Simple Sequence Repeats (SSRs) Analysis

High quality DNA is an essential requirement for developing DNA markers.

Therefore, DNA will be isolated and estimated from barley plants using a DNAeasy Plant

Mini Kit (Qiagen, Santa Clarita, CA).

Microsatellites or Simple Sequence Repeats (SSRs) are informative molecular

genetic markers in many crop species. SSR or microsatellites are short, tandemly

repeated DNA sequence motifs that consist of two to six nucleotide core units, and were

initially described in human (Litt and Lutty, 1989). They are highly abundant in

eukaryotic genomes and also occur in prokaryotes but at lower frequencies. These small

repetitive DNA sequences provide the basis for PCR-based multiallelic, co-dominant

genetic marker system.

a. SSR primers and SSR-PCR reactions

Three anchor microsatellite markers located on barley chromosomes were chosen from

GrainGenes database for analysis. These specific primers were synthesized by Operon

Biotechnologies, Inc., GmbH, Cologne, Germany.

SSR-PCR will be performed in 25 µl reaction mixture containing 1X PCR buffer, 1.5

mM MgCl2, 0.2 μM of each dNTPs, 1 μM of forward and reverse primers, 1U Go-Taq

Flexi polymerase (Promega) and 25ng genomic DNA.

b. SSR-thermocycling profile

PCR amplification will be performed in a Perkin-Elmer/Gene Amp PCR system 9700

(PE Applied Biosystem). A high stringency touchdown thermocycling profile will be

used as follows: an initial denaturation step for 5 min at 94ºC followed by 9 cycles (45 s

at 94ºC, 1min at 65ºC, 90 s at 72ºC). Then, 31 cycles of touchdown PCR will be



performed in which the annealing temperature was reduced by 1ºC every cycle until the

annealing temperature reached 56ºC and then a final extension cycle at 72ºC for 5 min

will be carried out.

c. Detection of SSR-PCR products

The amplification products will be resolved by electrophoresis in a 2 % agarose gel

containing ethidium bromide at 100 volts. SSR products will be visualized and

photographed using a Gel Doc™ XR+ System with Image Lab™ Software (Bio-Rad®).



Start Codon Targeted (SCoT) polymorphism analysis

The start codon targeted (SCoT) polymorphism is a novel, simple and reliable gene

targeted marker technique based on the translation start codon (Collard and Mackill,

2009). Primers for SCoT marker analysis were designed based on the conserved region

surrounding the translation initiation codon, ATG. Using a single 18-mer primer as a

forward and reverse primer in the PCR, Collard and Mackill (2009) designed thirty-six

primers that were used successfully for cultivar identification and genetic diversity

analysis in many crops.

a. SCoT primers and PCR reactions

Four SCoT primers were chosen from Collard and Mackill (2009) designed primers.

These primers were synthesized by Operon Biotechnologies, Inc., GmbH, Cologne,

Germany.

PCR will be carried out in 25 µl reaction mixture containing 1X PCR buffer, 1.5 mM

MgCl2, 0.2 μM of each dNTPs, 1 μM of primer, 1U Go-Taq Flexi polymerase (Promega)

and 25ng genomic DNA.

b. Thermocycling profile

PCR amplification will be performed in a Perkin-Elmer/Gene Amp PCR system

9700 (PE Applied Biosystem). The amplification will be programmed at 94ºC for 5 min

as an initial denaturation cycle, followed by 35 cycles, each cycle comprised of (94ºC for

1min, 50ºC for 1min, then 72ºC for 90 s) with a final extension at 72ºC for 7 min.

c. Detection of SCoT-PCR products

The amplification products will be resolved by electrophoresis in a 1.5% agarose

gel containing ethidium bromide (0.5ug/ml) in 1X TBE buffer. A 100 bp plus DNA

ladder will be used as molecular size standards. PCR products will be visualized on UV

light and photographed using a Gel Doc™ XR+ System with Image Lab™ Software

(Bio-Rad®).

Education

Practical handouts for The Regional Training Course on Utilization of Molecular Markers for PGRFA Characterization and Pre-Breeding for Climate Changes