Kew Angiosperm Genome Size Discussion Meeting and Workshop

Sponsored by Annals of Botany

9th - 12th September 1997


Kew Angiosperm Genome Size Discussion Meeting (11th-12th September 1997)

On 11-12th September 1997, 86 participants from 15 countries attended the Kew Angiosperm Genome Size Discussion Meeting hosted by RBG, Kew and sponsored by Annals of Botany. Sessions with 22 oral and 42 poster papers addressed aspects of genome size, including its systematic and ecological significance, intraspecific variation, and mechanisms of change.

Details of the discussion meeting are listed below:

Written papers from the meeting are published in a special issue of Annals of Botany volume 82 (supplement A, 1998).

Kew Angiosperm Genome Size Workshop (9-10th September 1997)

Prior to the discussion meeting, 19 scientists (from Argentina, Austria, Czech Republic, India, Italy, Mexico, New Zealand, UK and USA) attended the Kew Angiosperm Genome Size Workshop in the Jodrell Laboratory. They discussed best practice, identified knowledge gaps and made ten key recommendations for future research into plant DNA C-values. The group strongly endorsed the Angiosperm DNA C-values database, recommending that RBG, Kew continue to update this valuable information service.

Details of the workshop are listed below:


KEW ANGIOSPERM GENOME SIZE DISCUSSION MEETING

(11-12th September 1997)


Programme


11th September 1997

9.00 Introduction and Welcome Prof. Sir GT Prance FRS (Director of RBG Kew)
9.15 Current state of knowledge about angiosperm genome size  MD Bennett

Intraspecific variation

10.00 Intraspecific variation in genome size in angiosperms J Greilhuber 
11.10 Intraspecific variation in genome size in Helianthus J Price 
11.50  Intraspecific variation in genome size in Dactylis glomerata D Francis 
12.25  Detecting small differences in plant genomesize — sex chromosomes, Sesleria and bananas  J Dolezel 

Mechanisms of genome size change

2.00 Slipped strand mispairing and the evolution of genome size J Hancock
2.40 The basis, timing and possible significance of genome amplification in maize J Bennetzen 
3.20  Evolution and revolution in genome size JS Heslop-Harrison

12th September 1997

9.00 Introduction MD Bennett 

Genome size and systematics

9.05 Genome size variation and plant systematics D Ohri
9.40  Genome size evolution in the slipper orchids  T Cox 
10.00  Genome size and angiosperm phylogeny  IJ Leitch 
11.00 Genome size and reproductive development in angiosperms G Bharathan 
11.35 Nuclear DNA content and floral evolution in Silene sect. Elisanthe T Meagher
12.00 The phenomenon of genome size distribution in plant genera and its evolutionary implications RK Narayan
12.35  Genome size and orthoselection in angiosperms  PE Brandham

Ecological significance of genome size variation

2.10 Is genome size significant in plant ecology? P Grime
2.50 Genome size in weeds  L Hanson 
3.05  Genome size and environmental correlations in Zea mays  L Poggio 
3.55 Genome size, guard cell size and palaeobotany K Hunter 
4.15 Nuclear DNA amounts in gymnosperms B Murray 
4.50 Developing a rugged flow cytometer for plant genome research D Galbraith

Report on Kew Angiosperm Genome Size Workshop


Authors and titles of poster papers


1. Ainouche ML, Gourret JP, Misset MT. Genome size and evolution in the genus Bromus L. (Poaceae).

2. Bennett MD, Cox AV, Leitch IJ. Angiosperm DNA C-values database.

3. Bennett MD, Leitch IJ. Nuclear genome size in angiosperm plants.

4. Blackhall N, Anthony P, Davey MR, Power JB, Cocking EC. Flow cytometry for estimating angiosperm genome size.

5. Boon EJ. Flow cytometric analysis of plant nuclei stained with different DNA dyes compared with literature and to Feulgen densitometry.

6. Brandham PE. Genome size and orthoselection in angiosperms.

7. Cavallini AY, Ceccarelli M, Cremonini R, Frediani M, Natali L, Minelli S, Giordani T, Caceres ME, Maggini F, De Pace C, Durante M,. Cionini PG. Intraspecific genome size variations in plants: involved DNA sequences, effects on phenotype, roles in envionmental adaptation.

8. Cox AV, Abdelnour DG, Bennett MD, Leitch IJ. Genome size and karyotype evolution in the slipper orchids (Cypripedioideae: Orchidaceae).

9. Dolezel JY, Greilhuber J, Lucretti S, Lysák MA, Meister A, Obermayer R. Precision of plant DNA flow cytometry - inter-laboratory comparison.

10. Ebert I, Till W. Nuclear genome size in Pitcairnioideae (Bromeliaceae) with emphasis on the genus Pitcairnia.

11. Galbraith DW, Chytilova E, Lambert GM, Macas J. Nuclear targeting of GFP provides novel information about the plant genome.

12. Greilhuber J, Obermayer R. Cryptopolyploidy in Bunias revisited - quantitatively.

13. Grif VG, Punina EO. DNA amount per diploid genome variation in cytotaxonomy of angiosperm plants.

14. Hanson L. DNA amounts in angiosperm weeds.

15. Head J, Forster JW, Jenkins G. The molecular nature and cytological consequences of genome expansion in the Lolium / Festuca complex.

16. Hunter KL, Hunter RB. Guard cell size and water use parameters as related to genome size in modern and fossil samples of the North American desert shrub, Larrea tridentata.

17. Johnston JS, Bennett MD, Rayburn AL, Galbraith DW, Price HJ. Reference standards for determination of DNA content of plant nuclei.

18. Kiehn M. Nuclear DNA amounts of Rubiaceae.

19. König C. Nuclear genome size in polyploid Biscutella (Brassicaceae).

20. Kuipers AGJ, Kamstra SA, deJeu MJ, Ramanna MS, Heslop-Harrison JS, Jacobsen E. Unravelling the distribution of various types of repetitive DNA within the large genomes of Alstroemeria species.

21. Leitch IJ, Bennett MD. Genome size and angiosperm phylogeny.

22. Lim Y, Landi L, Mezzetti B, Leitch AR. Karyotype changes and loss of ribosomal DNA induced in the protoplast fusion product Raspberry x Blackberry.

23. Lysak M, Dolezelova M, Dolezel J. Flow cytometric analysis of nuclear genome size in bananas and plantains.

24. Marshall J, Hartman TPV, Blackhall NW, Power JB, Davey MR, Cocking, EC. Genomic in situ hybridization for plants with small genomes.

25. Mukherjee S. Genome size in angiosperms from different habitats.

26. Narayan RK, Khan MI, Mehetre SS, Khan MQ, Latif A. Genome size variation and evolution in Lathyrus, Nicotiana, Clarkia, Gossypium and Brassica.

27. ObermayerR, Greilhuber J, Swiecicki WK. Flow cytometric determination of genome size in new European Lupinus species.

28. Ohri D, Fritsch RM, Hanelt P. Evolution of genome size in Allium L. (Alliaceae).

29. Palomino G, Sousa M. Variation of nuclear DNA content in the biflorus species of Lonchocarpus (Leguminosae).

30. Papes D. Genome size and base composition of seven Quercus species; the relationship between chromosome number and DNA content variation.

31. Pearce S, Harrison G, Li D, Heslop-Harrison JS, Kumar A, Flavell AJ. Ty1-copia group retrotransposons in Vicia species.

32. Poggio L, Greizerstein E, Dopchiz L, Botini C, Ferrari MR, Naranjo CA. Nuclear genome size in several South American genera.

33. PoggioL, Rosato M, Chiavarino AM, Naranjo CA. B-chromosomes and alien cytoplasm as a cause of variation in nuclear genome size of maize.

34. PoggioL, Rosato M, Chiavarino AM, Naranjo CA. Nuclear genome size in Argentinian native races of maize.

35. Punina,EO, Grif VG. A correlation between DNA value and duration of mitotic cell cycle in Trilliaceae and relative families.

36. Reeves G, Francis D, Davies S, Rogers HJ. Hodkinson T. A correlation between genome size and altitude in natural populations of Dactylis glomerata.

37. Ricroch A, Brown SC. DNA base composition of Allium genomes with different chromosome numbers.

38. Samoylova TI, Meister A, Miséra S. The flow karyotype of Arabidopsis thaliana interphase chromosomes.

39. Schröder MB. Peak shifting - is DAPI-staining a reliable method for flow cytometry analysis of genome size in plants?

40. Schröder MB. Polysomaty in Asparagus officinalis L.

41. Suoniemi A, Kankanpä, J, Ylönen A, Schulman AH. Do retrotransposons contribute to genome size variation?

42. Yokoya K, Roberts AV, Lewis R, Mottley J, Brandham PE. Nuclear DNA amounts in roses.


KEW ANGIOSPERM GENOME SIZE WORKSHOP
(9-10th September 1997)


Participants


Prof. MD Bennett Jodrell Laboratory, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3DS UK

Dr G Bharathan Department of Ecology and Evolution State University of New York Stony Brook NY 11794 5245 USA

Prof. A Cavallini Dipt di Biologia delle Piante Agrarie, Sezione di Genetica Via Mattiotti 1/B Pisa I-56124 Italy

Dr AV Cox Jodrell Laboratory Royal Botanic Gardens Kew, Richmond, Surrey TW9 3DS UK

Dr J Dolezel Laboratory of Molecular Cytogenetics and Cytometry Inst of Experimental Botany Sokolovska 6, Olomouc CZ-77200 Czech Republic

Dr M Fay Jodrell Laboratory Royal Botanic Gardens Kew Richmond, Surrey UK

Dr DW Galbraith Dept of Plant Sciences University of Arizona Tucson 85721 USA

Dr J Greilhuber Institute fur Botanik der Universitat Wein Rennweg 14 Vienna A-1030 Austria

Ms L Hanson Jodrell Laboratory Royal Botanic Gardens Kew, Richmond, Surrey TW9 3DS UK

Dr J Johnston Texas A&M University Department of Entomology College Station, Texas 77843-2474 USA

Dr M Kiehn Institute of Botany & Botanical Garden University of Vienna , Rennweg 14 Vienna A-1030 Austria

Dr IJ Leitch Jodrell Laboratory Royal Botanic Gardens Kew, Richmond,Surrey TW9 3DS UK

Prof. B Murray School of Biological Sciences University of Auckland Private Bag 92019 Auckland New Zealand

Dr CA Naranjo Centro de Investigaciones Geneticas (UNLP-CONICET-CIC) CC 4 1836 Llavallol Buenos Aires Argentina

Dr RK Narayan University of Wales Aberystwyth Inst Biological Sciences, Sir George Stapledon Bld Penglais, Aberystwyth SY23 3DD, Wales UK

Dr D Ohri National Botanical Research Institute Rana Pratap Marg. PB No. 436 Lucknow 226 001 India

Dr G Palomino Lab. De Citogen., Jardin Botanico Instituto de Biologia Universidad Nacional Autonoma de Mexico (UNAM) DF 04510 Mexico

Prof. L Poggio Centro de Investigaciones Geneticas (UNLP-CONICET-CIC) CC 4, 1836 Llavallol Buenos Aires Argentina

Prof. HJ Price Texas A&M University Dept of Soil and Crop Sciences College Station, Texas 77843-2474 USA


The workshop programme


The programme was divided into six sessions. Each began with one or more invited mini-talk followed by a general discussion.

9th September 1997

Time Topic Co-chair Mini-talks
9.00 Welcome and introduction to the workshop  M Bennett 
9.15 Identifying and prioritising gaps in our knowledge of angiosperm genome size  D Ohri  1. What is known about angiosperm genome size and what isn't? (M Bennett) 
2. What is known about genome size in other plants? (B Murray) 
3. Gaps in our knowledge of genome size in regional floras (G Palomino - Mexico; D Ohri - India; C Naranjo - South America) 
Estimating C-values - best practice 
11.00  A. Calibration standards J Price 1. Are animal standards suitable calibration standards for estimating plant C-values? (J Price) 
2. Ideal plant calibration standards for estimating C-values (L Hanson) 
12.00 B. Feulgen microdensitometry J Price  1. Self-tanning and other staining problems with Feulgen microdensitometry (J Greilhuber) 
2.00 C. Flow cytometry  J Greilhuber  1. Flowing and glowing - Choice of fluorochromes (J Dolezel) 
2. Flowing and glowing - Practical issues for flow cytometry (S Johnston) 
3.00  D. Systematics J Greilhuber  1. Different classification systems (M Fay) 
2. Issues relating to systematics (I Leitch) 
4.00 E. Strategic/General/Quality control  J Dolezel 1. Strategic issues for global plant C-value research (M Bennett) 
2. Political and technical issues challenging C-value research (D Ohri) 
3. Developing a rugged flow cytometer for plant genome size research (D Galbraith) 

10th September 1997

Inter- and intra-specific variation
9.00 A. Intraspecific variation B Murray 1. Intraspecific variation as a technical artefact in genome size (J Greilhuber) 
2. The plastic genome (A Cavallini) 
3. Intraspecific variation in Zea mays (L Poggio)
B. Interspecific variation  B Murray  1. C-value doubling series in genome size (J Price) 
2. Lack of evidence for C-value doublings in Allium (D Ohri) 
11.00  Data Handling  I Leitch 1. Strategic issues and DNA databasing (T Cox) 
2. Options and issues for C-value data handling (I Leitch)
2.00 The significance of genome size variation J Price  1. C-values and reproduction (G Bharathan) 
2. The nucleotype (R Narayan) 
4.00  The future B Murray  1. Visions and issues (M Bennett) 
2. Future genome size science (J Price)


Outline summary report


Session I: "Identifying and Prioritising gaps in our knowledge of Angiosperm Genome Size"

A key aim of the workshop was to identify major gaps in our knowledge of plant DNA C-values and to recommend targets and priorities for new work to fill them by international collaboration. Presentations on regional floras, and analysis of representation of data in the Angiosperm DNA C-values database, all highlighted huge gaps in our knowledge, both in terms of the low numbers of species represented, but also in terms of systematic, life form, ecological and geographic representation. For example, a first DNA C-value estimate was still unavailable for the large majority (c. 68%) of angiosperm families.

Murray reviewed our knowledge of C-values in non-angiosperm plants where in some groups there were not 'intermittent gaps' but almost 'one continuous gap'. Representation was much better for gymnosperms than angiosperms, as values were published for c. 16% of gymnosperm species compared with c. 1% for angiosperms. The situation was worse for pteridophytes (c. 0.42%), and almost no C-value data were known for bryophytes (c. 0.1%), although locating data for these two groups had proved very difficult.

The workshop concluded that this level of ignorance was unsafe and unacceptable. New targeted work was essential to improve representation of both the angiosperm flora and of the other lesser-known plant groups. The difficulties encountered in locating DNA amount data for review at the workshop clearly demonstrated the value of user-friendly reference works. Thus, there was a clear need to bring together DNA amount data for species in other groups besides angiosperms, and make them easily accessible in one plant DNA C-values database.

Long-term and five-year targets were set. The ideal of a C-value for all taxa was unrealistic. However, estimates for 10-20% of angiosperms seemed both ultimately achievable and adequate for all conceivable uses provided these were carefully targeted to represent the various taxonomic groups, geographical regions, and life forms in the global flora. C-values for about 2,800 (c. 1%) angiosperm species had been estimated in the last 40 years. Two recommended five-year targets were agreed and set (see Key Recommendation 1). It was also strongly recommended that C-value data on both angiosperms and other non-angiosperm plant groups should be made readily available for reference purposes (Key Recommendation 2).

KEY RECOMMENDATION 1: To improve representation of data in the Angiosperm DNA C-values database by setting two five-year targets: 
  1. To estimate first C-values for the next 1% of angiosperm species (i.e. an additional 2,500 species). 
  2. To obtain at least one C-value estimate for a species in all angiosperm families. 

KEY RECOMMENDATION 2: Improve accessibility to plant C-value data by making them readily available for reference purposes as published reference lists and/or on the internet in one plant DNA C-values database. 


Session II: Best practice

A: Calibration standards

Calibration standards are of fundamental importance for accurate plant DNA C-value estimations. Indeed, many discrepancies in C-values reported for the same species were felt to probably reflect problems associated with the choice and/or use of calibration standards rather than genuine intraspecific variation.

Animal species for calibration standards
The inadequacies of chicken red blood cells (CRBC) as calibration standards for plants were highlighted. C-values for CRBC were reported to vary between authorities (up to 20%), sex (2-3%) and breeds. Moreover, CRBC showed different hydrolysis curves from plants. In view of these problems the workshop made the following key recommendation (Key Recommendation 3):

KEY RECOMMENDATION 3: Choice of calibration standards for estimating C-values in plants
In choosing calibration standards for estimating C-values in plants, animal cells, such as chicken red blood cells (CRBC) are not recommended. 

Plant species for calibration standards

The characteristics of ideal plant calibration standards were agreed to be as follows:

  1. Diploid (to minimise variation owing to aneuploidy).
  2. Single cultivars of a species.
  3. Easily available from more than one source.
  4. Stable.
  5. Suitable for both flow cytometry and Feulgen microdensitometry.
Following discussions, three plant species conforming to these criteria were recommended as calibration standards at the workshop (Key Recommendation 4):

KEY RECOMMENDATION 4: Recommended plant calibration standards
Three plant calibration standards were recommended for estimating C-values in plants: 
  1. Allium cepa cv. Ailsa Craig 
  2. Hordeum vulgare cv. Sultan 
  3. Pisum sativum cv. Minerva Maple 

B: Best practice - Feulgen microdensitometry

C: Best practice - Flow cytometry

Many aspects of the protocols for estimating DNA C-values using Feulgen microdensitometry and flow cytometry were discussed.

The following key recommendations of best practice (Key Recommendations 5) were agreed at the workshop.

KEY RECOMMENDATION 5: Recommended best practice for estimating DNA C-values 

Feulgen microdensitometry 

  1. Fixation of material: Formaldehyde fixation was recommended to avoid problems associated with tannins and other plant compounds.
  2. Feulgen stain: The workshop recommended that Feulgen stain should always be prepared from basic fuchsin powder and not bought as a pre-prepared Feulgen solution. The staining capacity of ready-made solutions of Feulgen were shown to reduce over time (weeks) once the bottle had been open. The quality of basic fuchsin powder varied between suppliers, reliable sources included Sigma and Merck.
  3. Pectinase treatment: While pectinase treatment can soften tissue, there is a risk that it can lead to loss of DNA if it contains DNase as an impurity. The following points were agreed: a) Only use pectinase if absolutely necessary, b) If used, perform pectinase digestion after staining not before and keep times as short as possible. c) Check source of pectinase to ensure it is free of DNase. 'Onozuka' pectinase from Yakult Chemical Company in Japan was recommended as a pure, reliable source of pectinase.
  4. Hydrolysis: Cold hydrolysis (i.e. 20oC 5N HCl or 25oC, 3M HCl) was strongly recommended over hot hydrolysis.
Flow cytometry 
  1. Fluorochome: The intercalating stain propidium iodide was recommended as the fluorochrome of choice at a concentration of 50 to 70 ppm.
  2. The buffer: The pH of the buffer should be between 7.2 and 7.4. Below pH 7.0 the nucleases act very rapidly leading to a loss of DNA.
  3. Chromosome counts: It was strongly recommended that chromosome counts should be made of material being measured by flow cytometry to check for ploidy level and chromosome aberrations which could cause variation in C-value estimates.

D: Best practice - Systematics

The group recognised the importance of accurate taxonomy for ensuring that DNA C-value data were attributed to the correct plant species. Thus it was strongly recommended that herbarium vouchers for wild plant species should be made for verification purposes and that they should be deposited in a recognised herbarium. It was also strongly recommended that researchers estimating C-values seek the collaboration of an expert taxonomist to help with identifications of their materials. These recommendations are summarised in Key Recommendation 6.

KEY RECOMMENDATION 6: To ensure that DNA C-value data are attributed to the correct plant species it is recommended that: 
  1. Herbarium vouchers are prepared of all wild plant species used to estimate DNA C-values and that these are deposited in a recognised Herbaria.
  2. When taxonomic identity of plant material is in doubt, researchers should seek the help of a competent taxonomist to ensure correct identification of the plant species. 

E: Best practice - Strategic/General and quality control

Strategic issues considered likely to affect the progress of plant DNA C-value research were discussed.

The 'obsolescence time bomb' threatening plant C-value research
A major factor likely to limit progress in plant C-value research seriously noted was the 'obsolescence time bomb' of ageing microdensitometers. Several workers noted that the equipment they used for Feulgen microdensitometry was considered obsolete and close to irreparable failure. For example, a high proportion of new C-value estimates were done on Vickers M85 microdensitometers made in the 1980s but now unsupported by the manufacturer. Without replacements there was already a serious risk that C-value estimation may cease in several countries, so preventing regional and global targets from being met. Specialist replacement microdensitometers, developed mainly for medical purposes, are expensive, probably prohibitively so especially for developing countries. Two alternative technologies considered were (i) flow cytometry, provided that a rugged, low-cost machine suited for conditions in developing countries became available, and (ii) computerised image analysis systems, although this seemed too expensive for most users.

Training and technology transfer
The essential need for training, and technology transfer was recognised, and it was decided to assess the demand and plan training workshops as necessary.

International collaboration
All participants agreed that international collaboration was essential to stimulate the research field and to obtain funding in an increasingly competitive market. Stressing the value of DNA C-value research in agriculture and biodiversity and conservation was considered as a possible route to increase the profile of genome research and thus to win increased funding.


Session III: Intra- and inter-specific variation

A: Intraspecific variation

The sometimes-contentious issue of intraspecific variation in DNA C-values was addressed and possible causes were discussed including taxonomic misidentification. The need to distinguish real variation from artefact was recognised. A reinvestigation of a few species claimed to show extreme intraspecific variation, such as in Colinsia verna and Poa annua, was suggested. To assist in evaluating suspected examples of intraspecific variation Key Recommendation 7 was proposed.

KEY RECOMMENDATION 7: Evaluating intraspecific variation: 

It was recommended that an informal mechanism to facilitate voluntary, independent tests and quality controls of C-value data showing intraspecific variation should be set up. Standardised growing conditions should be supplied with the seeds of plants being measured to try and overcome possible environmental influences. 

B:Interspecific variation

The possible existence of discontinuous DNA variation in DNA C-values within a genus and its origins was discussed. Problems associated with 'noise' in the database caused by several factors including poor taxonomy and technique were recognised as factors likely to add confusion to the debate. To help resolve the issue Key Recommendation 8 was agreed.

KEY RECOMMENDATION 8: To confirm or refute the existence of discontinuous distribution in DNA amounts within a genus: 

It was agreed that a set of species from genera claimed to show this phenomenon (e.g. Lathyrus, Nicotiana) should be re-measured together under uniform conditions to try and resolve the issue more conclusively. 


Session IV: Data Handling

The workshop was unanimous in agreeing that RBG, Kew had done an excellent job in establishing the Angiosperm DNA C-values database. Its future maintenance and possible improvements were discussed resulting in the recommendations below (Key Recommendation 9)

KEY RECOMMENDATION 9: Maintaining and updating the Angiosperm DNA C-values database: 
  1. RBG, Kew recommended to continue to manage the database. 
  2. Database to be extended to include other plant groups e.g. gymnosperms and pteridophytes.
  3. Database to be made relational to enable complex queries to be made.
  4. Database to include C-value data expressed in megabasepairs (Mbp) as well as picograms once conversion factor was agreed.

The possibility of submitting data electronically was raised and agreed that it would be useful although some form of quality control would be essential.


Session V: The significance of genome size variation

The significance of the huge variation in DNA C-values was discussed. It was noted that while over 100 different correlations concerning DNA amounts had been described the importance of evaluating these in the context of phylogeny was essential. Bharathan noted that some correlations concerning reproduction and DNA amount became insignificant when they were analysed within a phylogenetic context. New correlations for testing were suggested including those involving physiological factors (e.g. drought tolerance and DNA amount) and biodiversity and conservation issues (e.g. rates of extinction and DNA amounts). The importance of understanding the molecular mechanisms causing or associated with changes in DNA amount was stressed although it was noted that little work in this area was currently being done.


Session VI: The future

The meeting concluded with a wide-ranging discussion concerning the vision and issues for future DNA amount research. It was reaffirmed that DNA amount is a key biodiversity character and that it is essential to estimate C-values for at least an additional 1% of the global angiosperm flora to achieve improved taxonomic and geographical representation (see Key Recommendation 1). Target setting must use taxonomists' expertise to prioritise gaps and it was strongly recommended that this work be done in association with taxonomic institutions with large living and herbarium collections, such as RBG, Kew.

Collaboration should ideally involve international partnership (as previously agreed), and ensure that adequate training and technology transfer is achieved as an essential element of any collaborative programme. The workshop members are now the nucleus of such a group, and all interested parties will be invited to join.

Finally, the group agreed that it would be important to meet again and review progress (Key Recommendation 10).

KEY RECOMMENDATION 10: The future: 

A second Plant Genome Size Workshop and Discussion Meeting should be held in about five years time (c. 2002). 


The 10 Key recommendations from the Angiosperm Genome Size Workshop


  • KEY RECOMMENDATION 1: To improve representation of data in the Angiosperm DNA C-values database by setting two five-year targets:
    1. To estimate first C-values for the next 1% of angiosperm species (i.e. an additional 2,500 species).
    2. To obtain at least one C-value estimate for a species in all angiosperm families.
  • KEY RECOMMENDATION 2: Improve accessibility to plant C-value data by making them readily available for reference purposes as published reference lists and/or on the internet in one plant DNA C-values database.
  • KEY RECOMMENDATION 3: Choice of calibration standards for estimating C-values in plants.

  • In choosing calibration standards for estimating C-values in plants, animal cells, such as chicken red blood cells (CRBC) are not recommended.
  • KEY RECOMMENDATION 4: Recommended plant calibration standards.

  • Three plant calibration standards were initially recommended for estimating C-values in plants:
    1. Allium cepa cv. Ailsa Craig
    2. Hordeum vulgare cv. Sultan
    3. Pisum sativum cv. Minerva Maple
  • KEY RECOMMENDATION 5: Recommended best practice for estimating DNA C-values

  • Feulgen microdensitometry
    1. Fixation of material: Formaldehyde fixation was recommended to avoid problems associated with tannins and other plant compounds.
    2. Feulgen stain: The workshop recommended that Feulgen stain should always be prepared from basic fuchsin powder and not bought as a pre-prepared Feulgen solution. The staining capacity of ready-made solutions of Feulgen were shown to reduce over time (weeks) once the bottle had been open. The quality of basic fuchsin powder varied between suppliers, reliable sources included Sigma and Merck.
    3. Pectinase treatment: While pectinase treatment softens tissues, there is a risk that it can lead to loss of DNA if it contains DNase as an impurity. The following points were agreed: a) Only use pectinase if absolutely necessary, b) If used, perform pectinase digestion after staining not before and keep times as short as possible. c) Check source of pectinase to ensure it is free of DNase. 'Onozuka' pectinase from Yakult Chemical Company in Japan was recommended as a pure, reliable source of pectinase.
    4. Hydrolysis: Cold hydrolysis (i.e. 20oC 5N HCl or 25oC, 3M HCl) was strongly recommended over hot hydrolysis.
    Flow cytometry
    1. Fluorochome: The intercalating stain propidium iodide was recommended as the fluorochrome of choice at a concentration of 50 to 70 ppm.
    2. The buffer: The pH of the buffer should be between 7.2 and 7.4. Below pH 7.0 the nucleases act very rapidly leading to a loss of DNA.
    3. Chromosome counts: It was strongly recommended that chromosome counts should be made of material being measured by flow cytometry to check for ploidy level and chromosome aberrations which could cause variation in C-value estimates.
  • KEY RECOMMENDATION 6: To ensure that DNA C-value data are attributed to the correct plant species it is recommended that:
    1. Herbarium vouchers are prepared of all wild plant species used to estimate DNA C-values and that these are deposited in a recognised Herbaria.
    2. When taxonomic identity of plant material is in doubt, researchers should seek the help of a competent taxonomist to ensure correct identification of the plant species.
  • KEY RECOMMENDATION 7: Evaluating intraspecific variation:

  • It was recommended that an informal mechanism to facilitate voluntary independent tests and quality controls of C-value data showing intraspecific variation should be set up. Standardised growing conditions should be supplied with the seeds of plants being measured to try and overcome possible environmental influences.
  • KEY RECOMMENDATION 8: To confirm or refute the existence of discontinuous distribution in DNA amounts within a genus:

  • It was agreed that a set of species from genera claimed to show this phenomenon (e.g. Lathyrus, Nicotiana) should be re-measured together under uniform conditions to try and resolve the issue more conclusively.
  • KEY RECOMMENDATION 9: Maintaining and updating the Angiosperm DNA C-values database:
    1. RBG, Kew recommended to continue to manage the database.
    2. Database to be extended to include other plant groups e.g. gymnosperms and pteridophytes.
    3. Database to be made relational to enable complex queries to be made of the data.
    4. Database to include C-value data expressed in megabasepairs (Mbp) as well as picograms once conversion factor was agreed.
  • KEY RECOMMENDATION 10: The future:

  • A second Plant genome size workshop and discussion meeting should be held in about five years time (c. 2002).