Abiotic stress investigation methods of horticultural plants

cDNA - AFLP TECHNICAmplified Fragment Length Polymorphisms

(Vos et al., 1995)

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INTRODUCTION

According to the World Atlas of Desertification, UNEP (United Nations Environment Programme), dry lands cover 40% of the world’s land surface (almost 5.1 billion ha), and they are the habitat and source of livelihood for about 1 billion people. With the growth of population and the development of modern agro-industry and the other industries, extension of the dry land surfaces becomes a growing concern all over the world. This concern is reflected in the Convention on Biological Diversity’s dry lands work program, and in the establishment of the UN Convention to Combat Desertification. To mitigate the effects of drought, it is very important first to promote the effective and efficient use of existing plant resources, second to investigate the physiological processes and mechanisms that help plants to acclimatize to the water deficiency conditions. It is assumed that molecules and compounds such as proline, glycine betaine and soluble sugars etc. synthesized and accumulated during desiccation play an important role in the protection of the plants from stress . However, at this time the molecular basis for these protective mechanisms is almost unknown. In this experiment, the screening of the differentially expressed genes in water deficiency growth conditions was accomplished by means of the high reproductive technique (cDNA-AFLP). By this method we identified in stressed plants multiple polymorphic transcript-derived fragments (TDFs) that highlight specific plant reactions on molecular level.

MATERIAL AND METHODS

Plant materialRaspberry (Rubus idaeus L.) plants were grown in the greenhouse of the “V. Adavachi” Research Station (University of Agricultural Sciences and Veterinary Medicine of Iasi). Stressed plants were grown on the soil with 35% water hydration, and control plants were normal hydrated. Raspberry leaves were collected from mature greenhouse-grown plants and were immediately frozen in liquid nitrogen and stored at −80 oC.

RNA extractionTotal RNA was isolated from raspberry very fine grinded leaves, in liquid nitrogen, with the SpectrumPlantTotal RNA Kit according to the manufacturer’s protocol. RNA quality was checked using Bioanalyzer 2100 and RNA 6000 Nano Kit that allow visualization of 18S and 28S subunits and calculation of RNA Integrity Number (RIN)

Second-strand cDNA synthesisThe second-strand cDNA synthesis was performed with the SuperScript Double-Stranded cDNA Synthesis Kit that contains all of the reagents, except an oligo(dT) containing primer, necessary to make double-stranded cDNA from total RNA or poly A+ selected RNA (mRNA).

First-strand cDNA synthesisThe first-strand cDNA synthesis was accomplished with SuperScript II Reverse Transcriptase (RT) Kit that is an engineered version of MMLV RT with reduced RNase H activity and increased thermal stability.

AAAAATTTTTRT

AAAAATTTTTRT

RTAAAAATTTTT

The next steps of cDNA-AFLP analysis were made with AFLP Analysis System I, AFLP Starter Primer Kit according to the manufacturer’s protocols.Restriction Endonuclease Digestion To prepare an AFLP template, cDNA is 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).

Ligation of AdaptersAfter heat inactivation of the restriction endonucleases, the cDNA fragments are ligated to EcoR I and Mse I adapters to generate template DNA for amplification. These common adapter sequences flanking variable cDNA 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.

Amplification Reactions PCR was performed in two consecutive steps. In the first step, called preamplification, cDNAs are amplified with AFLP primers each having one selective nucleotide. The PCR products of the preamplification reaction was diluted and used as a template for the selective amplification using two AFLP primers (the EcoR I selective primer and Mse I selective primer), each containing three selective nucleotides. This two-step amplification strategy generated enough templates DNA for thousands of AFLP reactions. 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 × 109 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.

PCR preamplification reactions program performed 20 cycles at: 94oC for 30 s56oC for 60 s72oC for 60 s

EcoRI PRE-SELECTIVE PRIMER

MseI PRE-SELECTIVE PRIMER

GTAGACTGCGTACC AATT CA

CA AT GAGTCCTGAGTA

PCR selective amplification reactions program performed 33 cycles at:A. One cycle at 94°C for 30 s; 65°C for 30 s; and 72°C for 60 s;B. Lowered the annealing temperature each cycle 1°C during 9 cycles. This gives a touchdown phase of 10 cycles;C. The last 23 cycles at:94°C for 30 s;56°C for 30 s;72°C for 60 s.

Separation of Amplified Fragments on Denaturing Polyacrylamide GelsProducts from the selective amplification was separated on a 4% agarose gel. The resultant banding pattern was manually analyzed for polymorphic transcript-derived fragments (TDFs).

SELECTIVE PRIMER

GTAGACTGCGTACC AATT CACT

GACA AT GAGTCCTGAGTA

GTAGACTGCGTACC AATT CA

CA AT GAGTCCTGAGTA

EcoRI SELECTIVE PRIMER (labeled)

MseI SELECTIVE PRIMER

FAM

06.10.2011/Amplificare Selectiva probele 109 si 11535% hidratare (109-stres, 115 -martor) – Opal - sol/turba

0 Ladder

1 109E-ACC / M-CAG

2 115

3 109E-ACC / M-CAC

4 115

5 109E-ACC / M-CAT

6 115

7 109E-ACC / M-CTT

8 115

9 109E-ACC / M-CTG

10 115

11 109E-ACT / M-CAA

12 115

13 109E-ACT / M-CAC

14 115

0 Ladder

15 109E-ACT / M-CTA

16 115

17 109E-ACT / M-CAT

18 115

19 109E-ACT / M-CTC

20 115

21 109E-ACT / M-CTG

22 115

23 109E-ACT / M-CAG

24 115

25 109E-AAG / M-CAG

26 115

27 109E-AAG / M-CAA

28 115

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 15 16 17 18 19 20 21 22 23 24 25 26 27 28

RESULTS

0 29 30 31 32 33 34 35 36 37 38 39 40 41 42

0 43 44 45 46 47 48 49 50

06.10.2011/Amplificare Selectiva probele 109 si 11535% hidratare (109-stres, 115 -martor) – Opal - sol/turba

0 Ladder

29 109E-AAG / M-CAC

30 115

31 109E-AAG / M-CAT

32 115

33 109E-AAG / M-CTC

34 115

35 109E-AAG / M-CTT

36 115

37 109E-AGG / M-CAT

38 115

39 109E-AGG / M-CAA

40 115

41 109E-AGG / M-CTA

42 115

0 Ladder

43 109E-AGG / M-CTC

44 115

45 109E-AGG / M-CTT

46 115

47 109E-ACA / M-CTA

48 115

49 109E-ACA / M-CAT

50 115

07.10.2011/Amplificare Selectiva probele 136 si 13735% hidratare (136-stres, 137-martor) – Cayuga - sol/turba

0 Ladder

1 136E-ACC / M-CAG

2 137

3 136E-ACC / M-CAC

4 137

5 136E-ACC / M-CAT

6 137

7 136E-ACC / M-CTT

8 137

9 136E-ACC / M-CTG

10 137

11 136E-ACT / M-CAA

12 137

13 136E-ACT / M-CAC

14 137

0 Ladder

15 136E-ACT / M-CTA

16 137

17 136E-ACT / M-CAT

18 137

19 136E-ACT / M-CTC

20 137

21 136E-ACT / M-CTG

22 137

23 136E-ACT / M-CAG

24 137

25 136E-AAG / M-CAG

26 137

27 136E-AAG / M-CAA

28 137

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

0 15 16 17 18 19 20 21 22 23 24 25 26 27 28

07.10.2011/Amplificare Selectiva probele 136 si 13735% hidratare (136-martor, 137-stres) – Cayuga - sol/turba

0 Ladder

29 136E-AAG / M-CAC

30 137

31 136E-AAG / M-CAT

32 137

33 136E-AAG / M-CTC

34 137

35 136E-AAG / M-CTT

36 137

37 136E-AGG / M-CAT

38 137

39 136E-AGG / M-CAA

40 137

41 136E-AGG / M-CTA

42 137

0 Ladder

43 136E-AGG / M-CTC

44 137

45 136E-AGG / M-CTT

46 137

47 136E-ACA / M-CTA

48 137

49 136E-ACA / M-CAT

50 137

0 29 30 31 32 33 34 35 36 37 38 39 40 41 42

0 43 44 45 46 47 48 49 50

CONCLUSIONS

Screening 64 primer combinations identified multiple transcript-derived fragments (TDFs) that are differentially expressed in water stress conditions. The differences between control and stressed plants were qualitative when TDFs were either present or absent or quantitative when TDFs showed different levels of expression. The results show that cDNA-AFLP is a valuable technique for studying expression patterns of genes involved in sensitivity/tolerance mechanisms to water stress in red raspberry

THANK YOU

AcknowledgmentsThis study has been financed by the National Authority for Scientific Research, Operational Program POSCCE, ID 524, contract no. 151/2010, project title “The

significance of the relationship among genomic response, phenylpropanoid metabolism and photosynthesis for the optimization of biosynthetic potential of

raspberry and blackberry cultivars in abiotic stress conditions”

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