Issr markers may help specialy in defection of hybrids, ITSand ITS2 help in phylogeny and flow cytometry in ploidy level

Hi there,

I am actually having a similar situation with my study species, in which microsatellite genotyping and nuclear DNA sequences do not show any variation among individuals collected from different populations although floral patterns and leaf size show obvious differences. To make matters worse, the species might be a polyploid. Recently i thought genome-wide dominant markers (AFLP, RAPD, ISSR, etc) might shed some light on their variation aside from being fairly low-cost. I am still new in this but i hope my suggestion helps.

Same in mine (North American perennial Atriplex)

Hi, I use ISSR to distinguish even between half-brothers accessions. They are useful because you can be obtain many polymorphic bands. Try to probe primers di nt with 5' anchored, there are more polymorphic that 3'. To increase the specificity, use tri or tetra nt. Good luck.

you have to check a lot of primer combinations and ISSR markers to get the polymorphism. Some times it becomes hard to get the polymorphism by using less primer combinations.

If you are thinking that ISSR is not working, then try some other markers like RAPD, SSR or CAPS

Since polyploidy could be behind the phenotypic differences between these entities, I'd start there. And since they're not distinguished at genetic loci, recent divergence and/or ongoing gene flow are likely, so if it's not ploidy, then loci linked to adaptive phenotypes are going to be your best bet for finding differences (i.e. only a small proportion of markers). However, you need to be careful when using these to study gene flow in hybrid zones or build phylogenies.

Have you tried SNP markers?

i also suggest u ISSR markers......in my lab some of my seniors worked with issr markers only for inter specific hybrids in rice

hi use SRAP (sequence related amplified polymorphism) this is more accurate than ISSR and easily scorable bands.Also, since it targets ORF,it can be used to distinguish between itra and inter specific hybrids.

Very interesting information is sequencing data (cpDNA, etc.).

What exactly do you mean by tradition markers ?

what primer combinations or techniques have you used ?

Gene Tilling is the most efficient in Addition SNP with maximum primers can be use ful in mutants the chimera reverse resembling untreated controls Gene tilling gives very satisfactory results we can try vcj From Bangalore University Bangalore India

This is really on the border of my expertise, but have you considered RADSeq technology? It's really for population genetics but if you are having trouble distinguishing species this may be an economic way to do it.

Thank you very much for your answers! Maybe, I would try to apply ISSR and SNP analysis in my further work.

To Julian Gough: I know that RADSeq technology have surpasses AFLP and RFLP methods in many parameters, but I know nobody scientists who experienced in this technology in Ukraine. If you be so kind and can to advise such scientific laboratories would be interested in conducting such experiments with plant matherial, we are also ready to collaborate and try to apply this method too.

please could you precise your question ?

by classical markers do you mean cpDNA intergenic spacers, rDNA ITS etc ?

And by "phylogenetic" do you mean that you have more precise hypothesis about the existence of different species ?

As I understand your question and your reply about RAD sequencing your are rather in the realm of population genetics, perhaps phylogeography but I am not certain.

Before to choose a method you have to focus on your question.

I also suggest you can go for ISSR, SSR and SNP markers for your study. All d best

and my one request to Dr. Julian Gough, it will more useful if you can provide more information on RADSeq technology. It sounds very interesting. It would be of great help for the beginners if you could share some links or reprints in this regards.

To Alex Baumel:

By classical markers I mean cpDNA genes and intergenic spacers and rDNA ITS and by by "phylogenetic" I mean more precise hypothesis about the existence of different species

OK

Did you sampe enough intergenic cpDNA spacers ? if yes you have no phylogeographic structure, therefore true, well isolated species are less likely. But isolated populations, geographically or ecologically is still an hypothesis, you first have to well document occurrences and ecological data to support the existence of ecotypic, then you can use AFLP to test for a genetic isolation and the existence of different ESU.

From my point of view your question is more relevant about ESU (see Moritz 2002 or Crandall 2000) than to systematics.

Of course RAD sequecing is more an up to date method than AFLP, but currentely limits and pitfalls are not well known.

I think, for beginning you can try ISSR and SNP and SSR, in the cause that SSR-markers exists for Betula.

To Alex Baumel:

How many and what of intergenic cpDNA spacers we must to use to be able that it will be enough for our study? I'm afraid that we didn't analysed a lot of intergenic cpDNA spacers and thats why we maybe now can't speak about phylogeographic structure now.

Thank you in advance.

Investigate the variation within 10 differents intergenic spacers and use always sister species as an outgroup.

Could the nuclear DNA makers be useful in such situation? If these species were all good morphological species, I guess there should be some nuclear DNA makers, besides SSR, to distinguish them.

I used to work with wild potato species and we had events of ancient auto and allopolyploidization, and on top of that interspecific hybridization (at least with the species I used to work). We found out that sequence data from COS II (Conserved ortholog markers) were the ones that gave us the best results. If you are interested check these papers.

Wu F, et al. Genetics. 2006. 174(3):1407-20

Rodriguez, et al. BMC Evolutionary Biology. 2009. 9:191

Fajardo and Spooner. Systematic Botany. 2011. 36(1):163–170

My collaborators and I had the same problem (remarkable morphological differences without the expected molecular differences) using different families of plants, one genus within Verbenaceae (Lippia) and two genera within Bromeliaceae (Dickia and Encholirium). It seems very common in tropical environments (at least in Brazil).

My next try is to use microsatellites primers in order to investigate gene flow and other evolutionary processes. In this way, we will consider each genus of Bromeliacea (or even both) as a single lineage. I expect to detect several events of recente gene flow and show that morphological changes were very fast within this plants.

I think that we must change the tools in order to study plant phylogenies. Polyploids, dna changes between chloroplast, mitochondria and nucleus should impact negatively the phylogenetic results, which means that the interpretation of phylogenetic results must be different when one is studying animals or plants.

You can try a novel genetic marker, cDNA of progesterone 5 beta reductase, highly effective in many different plant familes such as genus Digitalis from Plantaginacae and very complex groups of Brasicacea, which is genus Erysimum. I am going with genus Digitalis monograph using this enzyme. see the article:

Herl V, Albach DC, Müller-Uri F, Bräuchler C, Heubl G, Kreis W (2008) Using progesterone 5β-reductase, a gene encoding a key enzyme in the cardenolide biosynthesis, to infer the phylogeny of the genus Digitalis. Plant Syst. Evol. 271: 65-78

RNase P RNA can be another phylogenetic marker gene to try for identification of evolutionary relationships. See (freely available) reference by Yanglong Zhu, Dileep K Pulukkunat, Yong Li, "Deciphering RNA structural diversity and systematic phylogeny from microbial metagenomes" Nucleic acids research. 01/2007; 35(7):2283-94. We can discuss whatever issues that may arise if you want to try this approach.

Good luck.

Hi,

For I am working actually on betula, I can give you several clues:

-There is hybrids, see http://link.springer.com/article/10.1023%2FA%3A1025063123552?LI=true for more info, then:

-Cp DNA is not suitable because it may come from one speicies to another, and depend more on location than species.

-Anonymous marker, are not suitable, bc they also come throug species, even if they do not share a same genome....

-Barcoding is pointless too

Best way to describe species is cytogenetic, you can find easilly the chromosome number, identify the genome in is harder, Fish/gish methode hasn't been successfull on Betula... you may ask to the autor of the paper I quote for some idea, She has curently a Betula project running... (just don't tell about me I should be working much more on it)

Hi,

Cp DNA analysis will not be useful if there is random outcrossing between the taxa . It is therefore advised to check other markers based on nuclear genome. We are very successful using Sequence Related amplified Polymorphism (SRAP) markers. This is working woderfully well. You can also use EST PCR RFLP of nucklear genome. This works very well even in species with low number of SNPs.

If interested you can check our papers on google

SRAP markers in coffee

EST PCR RFLP analysis in coffee

all are open access.

Otherwise pl contact me at

[email protected] .

I hope this may solve your problems

Ok, I don't know whether this will help you or not...we use microsatellite loci in jackfruit..see this: Witherup, C, D. Ragone, T. Wiesner-Hanks, B. Irish, B. Scheffler, S. Simpson, F. Zee, M. I. Zuberi, N.J.C. Zerega. In review. Development of microsatellite loci in Artocarpus altilis (Moraceae) and cross-amplification in congeneric species. Applications in Plant Science.

Do not use plastid DNA, use nuclear DNA, see e.g. Van den heede et al. 2003 Amer. J. Bot. 90: 481-495.

Recently, several papers have suggested that the markers with quick gene flow is suitable for species discrimination while the markers with low gene flow is suitable for trace the demorgraphic history of species, e.g. introgression. For your question i suggest you choose nuclear markers with low copy.

RJ Petit, L Excoffier Gene flow and species delimitation Trends in Ecology & Evolution, 2009

FK DU, RJ PETIT, JQ LIU More introgression with less gene flow: chloroplast vs. mitochondrial DNA in the Picea asperata complex in China, and comparison with other Conifers Molecular Ecology, 2009

I doubt that there are _many_ different Betula species in your part of the range. Check your species concept before trying any of these markers - if the taxa you are seeing are not real species (but merely geographical, morphological, ecological or ploidy variants), you won't see genetic differences with any of the recommended markers - because these differences simply do not exist. We have seen too many examples of that. I am refering to exaggerated botanical 'splitting', where the real species are rather 'lumped'.

Check polymorphic markers after RAPD gel analysis using parental genomic DNA then you can compare with Hybrids for the confirmation genomic DNA sequences appropriately SSR.

the alternative you have to confirm preparing karyotypic analysis first, for the genetypic variance at chromosomal level then the meiotic configaration will help you to check the ploidy level and phenotypic variability etc this preliminary work support you to further confirmation of molecular sequence both in parents and the Hybrids.

hI Tarieiev,

I would suggest you to look for the gene of Internal transcribed spacer (ITS). This gene comprised of two variable regions i.e. first and second internal transcribed spacer; separated by a conserved gene on 5.8S (ITS1-5.8S-ITS2).

This gene marker had been proved to be sensitive enough to differentiate not only pseudo-cryptic species but also cryptic species in most of the phytoplanktons, higher plant, fungi and even insects. Their secondary structures is relatively conserved throughout the eukaryotes and the compensatory base changes is considered another useful evidence to support the biological species concept with as high of 93%. You can understand more by reading papers by Coleman.

This is the paper that we recently published by using ITS sequence-structure information for marine diatom.

https://www.researchgate.net/publication/229731713_Morphology_and_molecular_characterization_of_Pseudo-nitzschia_(Bacillariophyceae)_from_Malaysian_Borneo_including_the_new_species_Pseudo-nitzschia_circumpora_sp._nov

Cheers, HongChang

Article Morphology and molecular characterization of Pseudo-nitzschi...

Hi Andrii,

I am using GPDH (nuclear marker) on roses but the variation is still low. If you just want to distinguish closely related species I read some papers using AFLPs (http://www.bioone.org/doi/pdf/10.1043/06-69.1) or microsatellites. However, I don't think these methods will help you to reconstruct the phylogeny. But if you have the money, you could think about next-gen. sequencing techniques (like RAD sequences: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037135).

Best of luck!

Maybe you'd like have a look at my paper about rhododendron interspecies hybridization. I did use cpDNA gene because I want to find which is the maternal parent. I used ITS because it is easy to amplify. But it is not good for analysis because of so called concert evolution. I suggest low copy nuclear gene and AFLP marker for this purpose. good luck.

Consider that the best results can be obtained by increasing as much as you can the coverage of the genome (i.e. include different loci). If possible try to sample more than one plant per assumed species to be sure of recover a species tree and not a gene trees (see the work of Lacey Knowles). Recentlty we have obtained a very well resolved phylogeny of Caribbean orchids. We used the nuclear ITS region and the plastid trnL-F region for the phylogenetic analyses. The former is biparentally inherited and is considered the fastest evolving sequence region in the plant genome (Álvarez and Wendel 2003; Hughes et al.2006). It has been shown to be of great utility for phylogenetic analysis among closely related species of orchids (van den Berg et al. 2005;Gustafsson et al. 2010) and flowering plants in general (Baldwin 1992; Baldwin et al. 1995). The trnL-F region has also been shown to be informative among closely related species (Wikstro¨m et al. 1999) and is widely used in orchids as well (van den Berg et al. 2000, 2005, 2009;Kennedy and Watson 2010; Monteiro et al. 2010), albeit less variable than ITS. You can find these references in my paper in Systematic Botany, available in my Researchgate profile. Including markers with different level of variation will help you to resuelve your phylogeny at different levels. Good luck!

I am attaching two papers I find most helpful; Shaw et al: the tortoise and the hare II and III

Article The tortoise and the hare II: relative utility of 21 noncodi...

I am interested to hear how you get on. Please keep posting

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