Asmaa 1 final report 10 days mission

Final Report For 2nd Training Mission Program of: EgyptianFinnish-Project: "Enhancing Development of Water Use Efficient Crops & Production Methods to Dry and Saline Conditions" In collaboration with: "Development of Fodder Resources in SinaiProject", FCRI, ARC, Egypt From: 10 - 20 June, 2013

By: M.Sc. Student: Asmaa M. A. Abd El-Aziz CRD-Cell Research Department FCRI-Crops Research Institute ARC- Agricultural Research Center

Supervision: Prof. Oiva Niemel채inen Prof. Alan H. Schulman Prof. Elina Kiviharju Prof. Naglaa A. Ashry Dr. Ruslan Kalendar Dr. Magdy M. Mohamed

(MTT-Finland) (Helsinki Univ.-Finland) (MTT-Finland) (ARC-Egypt) (Helsinki Univ.-Finland) (ARC-Egypt)

Finland; June 20, 2013

Acknowledgments I wish to express my deep gratitude to the project “Enhancing Development of Water Use Efficient Crops & Production Methods to Dry and Saline Condition�

Who offered this opportunity through its activities. Thanks are extended to all project members of FCRI, HU and MTT for arranging the training program and supervising the practical work throughout the training period,

(2)

ď ś Content:

No

Item

Page

1 2 3 4 5 6

Introduction Background Training Mission Objectives Financier Training Mission-Program Used Materials Training Activities Carried Out During The Mission 6.1: DNA extraction from maize and barley samples using CTAB method 6.2: Measuring the concentration of DNA

4 5 5 5 5 6 6 8

6.3: Running test gel 6.4: Running PCR for DNA amplification for retro-markers detection 6.5: Running samples to detect retromarkers

9 10 11

7

Sent Materials to Egypt

12

8

References

13

9

Report Annexes

14

(3)

ď ś Introduction: In recent years a great efforts were exerted on using biotechnological methods in plant breeding that might have its positive impact on the development and acceleration of accurate and faster results of any breeding program; the present training program was designed to integrate modern biotechnology methods on the development of water use efficient genotypes to cope with the expected water shortage.

Retro-markers, based on retro transposable elements in the plant genome is a modern simple and cost efficient method to generate molecular markers related to agronomic traits. This method was chosen to develop retro-markers related to a valuable traits related to seed production in alfalfa and faba bean through the activities of the present research project.

A great portion of this project is directed to capacity building including training of young researchers, a fresh graduated student who have just started her MSc. Was selected to be introduced to the MTT-Helsinki University genomic lab to get familiar with the modern techniques in plant genomics, and to be trained on retromarkers for further work to be done at Cairo lab.

Nine alfalfa and fourteen faba bean genotypes were chosen for this study including Egyptian, Polish and Finnish varieties. At the first stage biodiversity among the studied genotypes will be studied and the suitable retro-markers will be generated. The markers will be used in the suitable mapping population for marker assessed breeding program.

(4)

 Background: 1. Activity 1.1: Development of a retrotransposon-based set of DNAmarkers for alfalfa.  Training Mission Objectives: Familiarize her with the current genomics methods used at the MTT/University of Helsinki Plant Genomics Laboratory. Follow and participate to the alfalfa retrotransposon marker development carried out by Ph.D. student Marwa Ghonaim (FCRI) in her 2-months training period.  Financier Training Mission-Program: Egyptian-Finnish-Project:"Enhancing Development of Water Use Efficient Crops & Production Methods to Dry and Saline Conditions"; funded by: Ministry for Foreign Affairs of Finland.  Used materials: The following plant material of alfalfa were used in the training program, eight Egyptian genotypes in addition to one introduction, detailed description of the used material is listed in table one. Seeds of all material were germinated in the growth champers and used for DNA extraction in subsequent experiments.

Table (1): List of used alfalfa genotypes: Sub-species

Type of Population

Populations

Country

1. Giza-1

Egypt

sativa

Variety

2. Ismailia-1 Egypt

sativa

Variety

3. Ismailia94

Egypt

sativa

Population

4. Sinai-1

Egypt

sativa

Population

5. Siwa

Egypt

sativa

Variety

6. Jenaan

USA

sativa

Variety

7. WL-528

USA

sativa

Variety

8. Siriver

Austr alia

sativa

Variety

9. Radius

Polan d

sativa x falcata

Variety

Type of Variety Nondormant Nondormant Nondormant Nondormant Nondormant Nondormant Nondormant Nondormant Dormant

Ploidy

Group

4 x

Mediterranean-type Populations.

4 x

Mediterranean-type Populations.

4 x

Mediterranean-type Populations.

4 x

Mediterranean-type Populations.

4 x

Mediterranean-type Populations.

4 x

Synthetic variety

4 x

Synthetic variety

4 x

Synthetic variety

4 x

Hybrid of sativa x falcatapopulations.

Some barley samples related to my own MSc. Study were also used in addition to maize samples.

(5)

 Training Activities Carried Out During The Mission: 1. DNA extraction from maize and barley samples using CTAB method as follows: The following protocol is designed for small and large tissue samples (tissue volume 100-200 μl).

CTAB method for DNA extraction protocol  CTAB solution: 1-2% CTAB, 2 M NaCl, 10 mM Na3EDTA, 50 mM HEPES, pH ∼5.3; 100 ml: 1-2 g CTAB, 1.2 g HEPES-acid, 2 ml 0.5 M Na3EDTA, 40 ml 5 M NaCl;  Chloroform-isoamyl alcohol mix (24:1);  100% isopropanol (isopropyl alcohol, 2-propanol);  70% ethanol;  Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0).

1.

2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz. 2.

In 2 ml tube with mechanically disrupted seeds or leaves or herbarium or blood or DNA solution (CTABpurification) add fresh 1 ml CTAB solution buffer with RNAse A (the sample volume should not exceed 20% of lysis buffer), vortex very well and incubate the samples at 60-65°C during 30-60 min.

3.

Add 700 µl of chloroform, vortex very well (in the MM300 Mixer Mill for 1 min at 30 Hz); optionally: incubate the samples at 60-65°C during 30 min.

4.

Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube.

5.

Add 700 µl of chloroform, vortex very well for 1 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz).

6.

Spin at maximum speed in a microcentrifuge for 5 minutes.

7.

Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains of 800 µl 2-propanol, vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 3 minutes.

8.

Discard supernatant and wash pellet by adding 1.5 ml 70% EtOH, vortex well. Centrifuge at 14,000 rpm for 2 min and discard ethanol.

9.

The DNA pellet do not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.

Guanidine thiocyanate method for DNA extraction protocol  GuTC extraction buffer: 2 M guanidine thiocyanate, 10 mM Na 3EDTA, 50 mM HEPES, pH ∼5.3; the final concentration of guanidine thiocyanate may need to optimized for certain plant/animals tissue from 0.5 to 4 M;

(6)



Chloroform-isoamyl alcohol mix (24:1);  100% isopropanol (isopropyl alcohol, 2-propanol);  70% ethanol;  Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0).

1.

2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz. 2.

Add fresh 1 ml GuTC solution buffer (the sample volume should not exceed 20% of lysis buffer), vortex very well and incubate the samples at 6065°C during 30-60 min.

3.

Add 700 µl of chloroform, vortex very well for 1 minute creating an emulsion. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube. Repeat this step.

4.

Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains of 800 µl 2-propanol, vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 3 minutes.

5.

Discard supernatant and wash pellet by adding 1.5 ml 70% EtOH, vortex well. Centrifuge at 14,000 rpm for 2 min and discard ethanol.

6.

The DNA pellet do not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.

CTAB method for DNA extraction protocol from herbarium  CTAB solution: 2% CTAB, 2 M NaCl, 10 mM Na3EDTA, 50 mM HEPES, pH ∼5.3;  DNA binding microspin column with a silica membrane (e.g., GeneJET Plasmid Miniprep or HiBind DNA) spin columns;  Chloroform-isoamyl alcohol mix (24:1);  100% isopropanol (isopropyl alcohol, 2-propanol);  Washing buffer 1: 4 M guanidine hydrochloride, 40% isopropanol;  Washing buffer 2: 20 mMNaCl, 2 mMTris-HCl, pH 7.5, 1 mM EDTA and 80% ethanol;  Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0-8.5).

1.

2 ml Eppendorf Safe-Lock microcentrifuge tube with herbarium sample and glass ball grind in the MM300 Mixer Mill for 10 min at 30 Hz. 2.

Add fresh 1 ml CTAB solution buffer, vortex very well and incubate the sample for 1 hour at 60-65°C. 3.

Add 700 µl of chloroform, vortex very well (in the MM300 Mixer Mill for 1 min at 30 Hz); and incubate the sample for 1 hour at 60-65°C. 4.

Spin at maximum speed in a microcentrifuge for 3 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube.

5.

Add 700 µl of chloroform, vortex very well for 1 minute creating an emulsion. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube.

(7)

6.

Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube and add an equal volume (900 µl) of 2-propanol, vortex well and transfer of the sample to a microspin column, placed in a 2 ml collection tube. Centrifuge the tubes in a microcentrifuge for 1 minutes. Discard both the 2 ml collection tube and the flow-througt liquid.

7.

Add 500 μl washing buffer 1 to column and centrifuge in a microcentrifuge for 1 minutes and discard the flow-througt liquid.

8.

Transfer column to a second collection tube and wash by adding 700 μl washing buffer 2. Centrifuge the tubes in a microcentrifuge for 1 minutes and discard the flow-througt liquid. Repeat this step.

9.

Centrifugate empty column 2 min at maximum speed to dry. Transfer column to a clean 1.5 ml tube. Apply 200 μl 1xTE buffer and incubate at at 55°C for 10 minutes. Centrifuge the tubes for 1 minutes to elute DNA. Repeat elution with additional 100-200 μl of 1xTE buffer.

Method

1- 2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz. 2- In 2 ml tube with mechanically disrupted seeds or leaves or herbarium or blood or DNA solution (CTABpurification) add fresh 1 ml CTAB solution buffer with RNAse A (the sample volume should not exceed 20% of lysis buffer), vortex very well and incubate the samples at 60-65°C during 30-60 min.

3- Add 700 µl of chloroform, vortex very well (in the MM300 Mixer Mill for 1 min at 30 Hz); optionally: incubate the samples at 60-65°C during 30 min.

4- Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube.

5- Add 700 µl of chloroform, vortex very well for 1 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz).

6- Spin at maximum speed in a microcentrifuge for 5 minutes.

7- Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains of 800 µl 2-propanol, vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 3 minutes.

8- Discard supernatant and wash pellet by adding 1.5 ml 70% EtOH, vortex well. Centrifuge at 14,000 rpm for 2 min and discard ethanol.

9- The DNA pellet do not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.

2. Measuring the concentration of DNA DNA concentration was measured in the extracted samples using spectrophotometer, the resulted concentrations for the used samples are

(8)

shown in table 2. The concentrations of the samples was adjusted to be used for further work.

Table 2: sample DNA concentration for the used barley and Maize samples Barley samples 7039 Bomi 26-2 1150 Maize Samples 3 4 5 7 9 10 11 12 16

Concentration ng/ul 20 90 33 15,5 Concentration ng/ul 19.8 30.1 63.2 22.5 28.6 15.5 30.4 35 26.3

3. Running test gel.

The extracted DNA from four barley samples were run on agrose gel to test its quality, the results is shown as follows; sample extraction was perfect and suitable to proceed with other steps.

(9)

4. Running PCR for DNA amplification for retro-markers detection.

DNA amplification for the extracted barley samples were done on Biometra thermo cycler apparatus. Sample preparation was achieved as follows: 16 µl of premix + 4 µl of DNA 5ng/µl The PCR protocol was achieved as followd: *95C – 2 min; *32 cycles : (95 – 20 sec, 60C - 30 sec, 72C – 90 sec) *last elongation 72C – 5 min.

The following list of primers were available and tested, from which I hereafter represnt four (marked in red for maize and in blue for naize). The DNA profiles of the samples are shown using iRAP peimers . seq_id

sequence

IRAP Efficiency

Source

Length

Tm(°C)

CG(%)

LC(%)

PQ(%)

4333

ccatggcgagcagatgtgct

3

67

20

60.7

60.0

84

80

4334

tatgttgggaaaacggggtg

5

54

20

55.9

50.0

76

73

4335

tgaaaatcgcagcactcctaggt

0

54

23

58.0

47.8

93

93

4336

tgtacaaagactggcacgagatgc

3

34

24

59.4

50.0

90

87

4337

tgcagcttcgacggccaagatgc

3

34

23

64.7

60.9

93

93

4338

ctttgccaatctgtgatgcag

1

21

21

55.9

47.6

87

87

4339

tcatacactgttaagtgctcaacc

1

21

24

55.1

41.7

90

89

4340

atggttgtcgaaactccagc

1

10

20

55.9

50.0

97

93

4341

gtcccacagcttgggcaacag

2

10

21

62.5

61.9

85

85

4342

gattgcaaagcctatttcgctg

2

1

22

56.2

45.5

90

84

4343

caattatcaaaccctccactttcg

2

1

24

55.2

41.7

83

73

4344

ctcttctcaaacaccaggagtc

0

1

22

55.9

50.0

82

80

4345

ccaaacaggatttctgaacgactc

0

1

24

56.9

45.8

90

73

4346

accttgctgctaaggttgaggcat

0

1

24

60.5

50.0

86

86

4347

ctgctccaaatccctttcttttgctccaaga

5

2

31

62.4

45.2

69

69

4348

ttagatgaaaccaacgatcccaaggct

4

2

27

59.6

44.4

89

89

4349

ctcaccccttctgcttggaaatgttgct

5

17

28

63.1

50.0

83

83

4350

gtaaataggctcccagggcatcaagccctc

4

17

30

66.1

56.7

81

80

4351

caggcaagaatgagcgtctc

3

55

20

57.1

55.0

89

88

4352

acccggaagggcggttcatgcaa

3

55

23

65.2

60.9

93

93

4353

gcaagtaactatatctggccaacc

1

55

24

56.4

45.8

86

86

4342 4343

gattgcaaagcctatttcgctg caattatcaaaccctccactttcg

2 2

1 1

22 24

56.2 55.2

45.5 41.7

90 83

84 73

4347

ctgctccaaatccctttcttttgctccaaga

5

2

31

62.4

45.2

69

69

4348

ttagatgaaaccaacgatcccaaggct 4

2

27

59.6

44.4

89

89

(10)

5. Running samples to detect retro-markers.

The extracted DNA was tested against the set of markers as mentioned above and a sample of DNA profiling using iRAP primers is shown bellow for Maize and alfalfa.

Testing the amplification on agarose gel was achieved (40 Âľl 1x loading buffer; mix well, and 8 Âľl was used for loading in gel.

Electrophoresis was done in with long gel 1.4% agarose (1xTHE buffer); 50V, ~14h

(11)

ď śSent materials to Egypt: Below is a list of sent materials to Cairo Lab. I brought this chemicals to start testing the protocol at Cairo Lab, other chemicals will be also sent company VWR BioNordika Thermo Fischer ImmunoDiagnostic City Labs (OmniPur)

ordered EZNA PLANT DNA MINI KIT, 50 tests, D3485-01 Guanidine thiocyanate (AppliChem Gmb; A4335,1000) Finnpipette 2 GLP-kit 1 kpl 0.5 kg ReSolute Wide Range Agarose TRIS, OmniPur 77-86-1, 500 g

(12)

References: 

Modified from Fedoroff, N.V. (1998). The discovery of transposable elements. In Discoveries in Plant Biology, vol. 1 (eds Shain-Dow Kung and Shang-Fa Yang), World Scientific Publishing Co. Pte. Ltd., Singapore, pp. 89–104.



R Kalendar1, AJ Flavell2, THN Ellis3, T Sjakste4, C Moisy1 and AH Schulman1,5 (2011).

Analysis of plant diversity with retrotransposon-based molecular markers. Heredity, 106, 520–530

(13)

Report Annexes:

(14)

(15)

(16)