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Sent: 13 June 2005 12:14
Subject: 43: Re: Establishing a universal molecular marker data base
I am Rajeev Varshney, from the Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.
I enjoyed reading several messages on 'Establishing a universal molecular marker data base'. In particular, I agree with the recent message of Marilyn Warburton (nr. 42, June 10) who mentioned that ALL MARKERS tested did not produce ESSENTIALLY SIMILAR results across the lab in their studies. In fact, we also had similar kind of experiences. However, I think that in comparison to genomic simple sequence repeat (SSR) markers, EST-derived SSR markers shower higher reproducibility as we get very strong band or distinct peak in genotypes of interest (see a recent review: R.K. Varshney, A. Graner and M.E. Sorrells. 2005. Genic microsatellite markers in plants: features and applications. Trends in Biotechnology 23:48-55 - copy on request from rajeev (at) ipk-gatersleben.de). [EST-SSRs are SSRs (also known as microsatellites) that have been identified from DNA sequences arising from expressed sequence tag (EST) sequencing projects. An advantage they have over traditional microsatellites is that they are present in expressed regions of the genome (i.e. the sequences are from the small fraction of the genome that contains genes, that result in proteins)...Moderator].
Furthermore, we argue that for molecular characterization of germplasm material (that needs to be verified or used across the labs)- single nucleotide polymorphism (SNP) markers are the best class of molecular markers. In the case of SNP markers, we expect (generally) one of two alleles and there should not be ambiguity in interpreting the results obtained by using SNP markers on any platform like Pyrosequencing etc. I am aware, though, of the fact that at present we are having a limited number of SNP markers in only a few crop plant species and that SNP genotyping (and genotyping platform) is very expensive. At the same time, accumulation of sequence data through genome/EST-sequencing projects in many plant species is providing us with the sequence data for more than one genotype for a given gene. Many bioinformatic tools are publicly available that can be used to identify the SNP (between two or more than two genotypes) and development of SNP markers. Furthermore, some bioinformatic tools like SNP2CAPS developed at IPK, are capable of identifying the restriction enzyme to assay a particular SNP. Thus cleaved amplified polymorphic sequence (CAPS) -based assays using agarose gel and restriction enzymes can be exploited for genotyping the SNP (in the form of 'cut' or 'uncut' fragment) in the germplasm collection.
Thus, it seems that probably SNP markers would prove to be the markers of choice for genotyping the genebank/breeding materials (and storing them in database) across the lab and we should expect many more markers with the possibility of high throughput genotyping in cost-effective manner.
Rajeev Kumar VARSHNEY, Ph.D.
Institute of Plant Genetics and Crop Plant Research (IPK)
D 06466 GATERSLEBEN
Tel: ++ 49 39482 5594(off.),5231 (lab) Fax: ++ 49 39482 5595
E-mail: rajeev (at) ipk-gatersleben.de / rajeevkvarshney (at) hotmail.com
Web : http://www.ipk-gatersleben.de/en/ ; http://pgrc.ipk-gatersleben.de/
[Firstly, as a refresher, Marilyn (Message 42) wrote that from their use of SSR markers in maize with partner labs in different countries "The main conclusion of these studies was that if different laboratories use the exact same protocols and platforms for genotyping, with multiple internal controls (same genotypes run in all labs), SOME of the markers (perhaps up to half) can be compared between laboratories".
Secondly, the abstract of the Varshney et al. 2005 paper referred to above is "Expressed sequence tag (EST) projects have generated a vast amount of publicly available sequence data from plant species; these data can be mined for simple sequence repeats (SSRs). These SSRs are useful as molecular markers because their development is inexpensive, they represent transcribed genes and a putative function can often be deduced by a homology search. Because they are derived from transcripts, they are useful for assaying the functional diversity in natural populations or germplasm collections. These markers are valuable because of their higher level of transferability to related species, and they can often be used as anchor markers for comparative mapping and evolutionary studies. They have been developed and mapped in several crop species and could prove useful for marker-assisted selection, especially when the markers reside in the genes responsible for a phenotypic trait. Applications and potential uses of EST-SSRs in plant genetics and breeding are discussed"...Moderator].
Sent: 13 June 2005 12:14
Subject: 44: Markers vs. morphology for diversity analysis
This is PK Gupta, again, from the CCS University, Meerut, India. We have been involved in molecular marker work in bread wheat for almost a decade. In particular, we developed and used simple sequence repeat (SSR) markers (both genomic SSRs and EST-SSRs) for a variety of studies including gene tagging, QTL analysis, diversity studies, transferability of SSRs, etc.. We are also surveying the repetitive and unique sequences of the genome for the organization of SSRs within the genome using methyl filtered and Cot fractionated sequences of wheat genome.
Regarding the use of molecular markers for diversity analysis, we felt discouraged when, after first using SSRs (published in Theoretical and Applied Genetics (TAG), 1999), we tried SAMPL (published in TAG, 2002) and AFLP on the same set of genotypes (published in Biochemical Genetics 2004). The three marker systems gave different results, suggesting that perhaps data on large number of morphological traits will still give the best estimate of diversity. This may look like a retrograde step, but when we pooled the data from all the three marker systems and used it for diversity analysis, the results were comparable with those obtained using about a dozen morphological traits. [Selective amplification of microsatellite polymorphic loci (SAMPL) analysis is an extension of Amplification Fragment Length Polymorphism (AFLP) technology to include amplification of microsatellite loci...Moderator].
We also argued that a set of SSR markers (about 20-50), if used for diversity analysis, samples a very small fraction of the genome, and cannot be representative of the whole genome, which should be scanned for diversity analysis. Consequently, even if several labs combine together, they need to understand the limitations of using molecular markers for diversity analysis. Perhaps, for characterization and identification or for DNA barcoding of the germplasm, DNA-based markers are OK. But, for diversity analysis, unless large number of markers from different marker systems and representing whole genome are used, one would not get a correct estimate of diversity. After having worked for a decade on a variety of molecular markers, I believe that maximum information from morphology will still give the best measure of diversity.
One should also recognize that many of the markers that we use belong to repetitive and non-coding regions of the genome, so that a selection should be made and functional markers should be used for analysis. This has been done in some studies, and we have published a review on functional markers and their utility (Functional and Integrative Genomics, 2004). Therefore, as an afterthought, we feel that even if markers need to be used, functional markers should be preferred over random genomic markers for diversity analysis.
Honorary Emeritus Professor and INSA Senior Scientist
Molecular Biology Laboratory
Department of Genetics and Plant Breeding
Ch. Charan Singh University
Tel (Lab): 91-121-2768195
TeleFax : 91-121-2768195
e-mail : pkgupta36 (at) yahoo.com
Sent: 13 June 2005 12:15
Subject: 45: Re: Molecular characterisation of animal genetic resources
I am Chanda Nimbkar from the Nimbkar Agricultural Research Institute, a small non-governmental organisation (NGO) in Maharashtra State of India. I am currently finishing a PhD in animal breeding at the University of New England in NSW, Australia. At our institute in India, we have an ongoing sheep breeding program which uses DNA testing to detect animals' genotypes within a month after birth so that this information can be used for early selection decisions. We work closely with local sheep owners and are testing improved genotypes in their flocks.
I have read with great interest, the important issues raised by Ilse Köhler-Rollefson (Message 31, June 9), Olivier Hanotte (Message 36, June 10) and Hans Lenstra (Message 35, June 10).
I would like to make a few comments in support of some of the pertinent issues Olivier Hanotte has brought into the debate. I see a worrying trend in developing countries such as India where it appears that a vast amount of effort and money are being put into molecular characterization while other areas more directly linked to poverty alleviation such as performance characterization of indigenous breeds leading to genetic improvement programs based on simple principles and methods of animal breeding have been ignored consistently. We need to ask ourselves whether the research funding in developing countries going into some areas of animal biotechnology is because of their 'glamour' and whether the scientists always keep in sight the final application of their research findings. e.g. tremendous efforts and money are invested in perfecting the techniques of embryo transfer (and even cloning) when hardly any attention is paid to producing highly genetically superior animals, for the multiplication of which these techniques were invented.
Ms. Chanda Nimbkar
Animal Science (Room 35, Postgrad Annexe)
School of Rural Science and Agriculture
University of New England
Armidale, NSW 2351
Phone: Work +61-2-6773-3008
cnimbka2 (at) une.edu.au
Sent: 13 June 2005 12:16
Subject: 46: DNA genebank - survey of institutions worldwide
This is Carmen de Vicente, again, and this message related to the DNA genebank issue.
About 2 and a half years ago, IPGRI organized a consultation among germplasm experts to talk about the future role of genebanks in the modern era of molecular genetics and genomics (http://www.ipgri.cgiar.org//publications/pubfile.asp?ID_PUB=986). One of the points discussed was DNA banking. As a result, in 2004 a questionnaire was circulated among 816 institutions worldwide (134 countries) to gather up-to-date information on plant genetic resources DNA banking activities and outlooks. The 816 institutions included both national and international institutions working with plant genetic resources, botanical gardens, universities and the private sector. 243 institutions replied and the survey showed that only 20% of those keep DNA as a genetic resource. Most of the replies mentioned lack of knowledge about the subject as well as budgetary constraints and lack of capacity (equipment and expertise). Most people requested information about protocols, standards, documentation, etc. but also about legal issues involved in exchange of DNA. The need for coordinating activities was also mentioned. IPGRI is at this moment finishing up a multi-authored publication, with the aim of establishing a point of departure by assembling information on different aspects related to DNA banking in general. Authors of the publication include some of the names who have been mentioned in this conference. I'm sure FAO will be happy to announce the publication when ready. [These are exactly the kind of publications included in the FAO-BiotechNews e-mail newsletter, posted roughly once a month - http://www.fao.org/biotech/news_list.asp?thexpand=1&cat=131 ...Moderator].
M Carmen de Vicente
International Plant Genetic Resources Institute (IPGRI),
Office for the Americas,
c/o CIAT, A.A. 6713, Cali,
c.devicente (at) cgiar.org
Sent: 13 June 2005 12:17
Subject: 47: DNA bank versus seed bank
This is D. Vijay from India, again.
I totally agree with Kioumars Ghamkhar (Message 34, June 10). I had a brief discussion about the use of DNA banks with Dr. A.D.H. (Tony) Brown during his recent visit to IPGRI, New Delhi Office. We came to a conclusion that even though there are lot of advantages for DNA banks compared to traditional seed banks, they cannot replace seed banks, at least in the near future. Already Kioumars has mentioned some advantages and disadvantages of DNA banks, but here I want to emphasize why they cannot replace the traditional seed banks in the near future:
- From seeds, entire plants can be regenerated which is not possible from the DNA stored.
- For the use of DNA as replacement to the live plant, we should know the sequence of it first. Sequencing all the existing species is a mega task and will take sufficient time and, once sequencing is successful, then comes the identification of genes.
- Thus, DNA bank is fully useful only when all the genes in a species are known, otherwise it can be used partially for the known genes. Identification of genes in various species is itself a very big task and needs lots of sophistication in the existing molecular techniques.
Even the Royal Botanic Gardens, Kew, who are having highest number of DNA samples (22,000) in their DNA bank, clearly mentioned on their website that "We thus make no claims about the quality or concentration of these samples nor how these samples can be used (degraded samples are not suitable for AFLP, for example)". This shows still there is a need to work on a foolproof method to get a high quality DNA from different kinds of tissues (?).
Thus, DNA banks are the technology for the future. They may not replace the seed banks in the near future but certainly can supplement them. To my knowledge, there are only two major DNA banks in the world with respect to plant species, one is 'Royal Botanic Gardens', Kew, and second one 'Nationaal Herbarium Nederlands', The Netherlands, both of which even supplies the DNA samples of the species available with them.
International Plant Genetic Resources Institute
IPGRI Office for South Asia
NASC complex, Pusa Campus
New Delhi - 110 012, India
E-mail: vijaydunna (at) gmail.com
[The Kew Gardens website for their DNA Bank Database (http://www.kew.org/data/dnaBank/homepage.html), informs that: "The RBG Kew DNA Bank contains over 22,000 samples of plant genomic DNA, all stored at -80°C. DNA is extracted from particular taxa of interest that are then databased with information on names, collectors, localities etc. Each sample is vouchered (made into a herbarium specimen). DNA samples are currently sent to collaborators all over world. It is our aim to open up this resource to a wider audience in the future. DNA samples can be purchased from Kew using this site. The fees assessed do not imply that DNA samples are being purchased, but rather this fee is meant to offset the cost of producing the sample, maintaining it and shipping it. The great majority of these DNA samples are of high molecular weight and reasonable concentration. Some as noted in the comments column are somewhat degraded because the tissue from which they were extracted was not dried properly, whereas others are more dilute than desirable, but some taxa never produce much DNA. We thus make no claims about the quality or concentration of these samples nor how these samples can be used (degraded samples are not suitable for AFLP, for example). Samples, unless otherwise noted in the comments column, have been cleaned on a caesium chloride/ethidium bromide gradient, so they are reasonably free of RNA and Taq-inhibitory chemistry, but in some cases further cleaning and concentration by means of a silica-based column may be necessary before amplification can be achieved. We are happy to advise about problems, and if degradation during shipment occurs we will replace such samples without further charge. The amount of DNA sent is suitable for PCR-based techniques (typically about 25 µl). If you require a greater amount of DNA, this may be possible, but you should contact us to discuss this (firstname.lastname@example.org)"...Moderator]
Sent: 13 June 2005 12:17
Subject: 48: Re: DNA genebank
This is Kioumars Ghamkhar from Australia, again.
Regarding message 39 (June 10) from Glaucia S.C. Buso: The only well-established DNA bank that I am aware of is in the Kew botanic Gardens in UK. There is also another DNA bank at the Trinity College (Dublin, Ireland). I guess there is a project in Brazil at Jardim Botanico (Botanical Garden) in Rio de Janeiro to establish a DNA bank, as well.
Criteria for long term preservation is keeping them in -80 or even -20 degrees, with fresh leaves in silica gel as a back up.
In terms of publications, there should be, but I do not have any in my mind at present. Searching the web is a good idea.
With regard to Rajeev Varshney's message (within nr. 39) on an Australian DNA bank, many thanks for introducing the centre. This is one of the advantages of this email conference. Yet, this bank is not a well-established one and even the Irish DNA bank is more developed than the Australian one.
Dr Kioumars Ghamkhar
Centre for Legumes in Mediterranean Agriculture (CLIMA)
University of Western Australia
35 Stirling Highway
Crawley WA 6009
Voice: 61 8 6488 7120
Fax: 61 8 6488 1140
E-mail: kioumars (at) cyllene.uwa.edu.au
Sent: 13 June 2005 12:18
Subject: 49: Re: Introducing biotechnology in rural areas = sausages to a new born baby
This is Vijay.D from India, again
Regarding Message 16 (June 8) and Message 29 (June 9) from Roger Djoulde Darman:
I agree completely with Carmen de Vicente (Message 27, June 9) and Kioumars Ghamkhar (Message 33, June 10). I want to elaborate it a little further. Here, we should understand that technology is not the problem but its adoption is the problem. Lab to Land is very crucial to make the technology useful for the common good. When the technology in the case of cassava fermentation has failed (as per Roger), it means we should check the reasons why it failed? (as emphasized by Carmen). If the complicated procedure itself made it a failure then one should work to develop an easier technology. For example, old world computers which are huge in size and too costly and are not so popular but the present scenario is completely different. How did this became possible? So with further research only the technologies developed can be made user-friendly and will one day reach the common man. Research and technologies are like stepping stones. A single technology may not yield every thing.
Yes, what Roger said is correct. PCR or NMR are not being used by rural persons but the possible output from them can be used in the right way for the benefit of the so-called common man. The best example is breeding. The farmer has nothing to do with the art and science of maintenance of numerous lines, crosses, testing for resistance in glass houses and in phytotrons, checking the dominance and recessiveness of characters etc. But the end result of these technologies is an improved variety or a new hybrid which will change his fate. The so-called green revolution which has changed the scenario of world food production, particularly in developing countries like India, is remarkable. But this has happened only when the farmers had adopted the new varieties. It did not happen all of a sudden or overnight, it took years for the farmers to accept the new improved lines.
Coming to the present topic: Use of modern biotechnology for the conservation and use of germplasm has its own relevance. With the drastic change in farm practices, which includes excessive monoculture, there is a need to preserve the existing diversity to use it for further development of existing populations. So, in the situation of dwindling on-farm maintaince of this naturally existing diversity, cryopreservation promises to a great extent.
Thus, the technology developed should be used properly with user friendly methodology along with adoption of proper extension techniques for its successful transfer from lab to land which should also be given proper emphasis. If it failed in one case (as Roger said in the case of cassava fermentation) it is not a permanent failure, it is only transitory. This shows that a more user-friendly methodology has to be developed but the basic technology that ferments the cassava remains the same. Here, we should understand the difference between technology and methodology used by the end user. The molecular markers, PCR etc. are technologies the end user does not use them - instead he will use the outcome of these technologies.
International Plant Genetic Resources Institute
IPGRI Office for South Asia
NASC complex, Pusa Campus
New Delhi - 110 012, India
E-mail: vijaydunna (at) gmail.com
Sent: 13 June 2005 12:19
Subject: 50: Learning a lot in this conference - Panama
My name is Jazmina Urriola and I am studying biotechnology at the Saint Martin University in Panama. I am currently attending the third year of the career and I would like to be a specialist in the agrobiotechnology field, which is quite interesting.
Here in Panama, agrobiotechnology is not as developed as in other developing countries. The state has not provided the financial support that is required, there are few well educated people and there is a lack of equipment and infrastructure. However, researchers are trying to take advantage of the resources available, focusing on the improvement of our main crops. They are conserving sweet potato and yuca, and are using micropropagation for pixvae and pineapple. They have already characterized two tubers, called otoe and name, and they have also worked with tomatoes, achieving excellent results. Panamenian researchers are also working with livestock. The results have been great!!! Actually, an agrobiotechnology laboratory was opened yesterday [June 10...Moderator] in order to help farmers and rural people, who have well accepted these new technologies.
Here in Panama there is a lot to do regarding to this topic. I am learning a lot in this conference and I would like to thank all the participants for sharing their experiences. I wish to be able to apply all this knowledge, in order to benefit my country, mainly the rural population.
Jazmina I. Urriola S.
Faculty of Biological Sciences
Universidad San Martin de Panama
jazmina_ius (at) yahoo.com