Kindly could anybody suggest any highly variable markers for discrimination for closely related plant species/lower taxonomic level identification. If DNA barcoding could able to go at that level? I have already used rbcla, matK, ITS2 , trnL-F and psbA-trnH but it didn't able to discriminate. Now, what should I use? any other highly variable intergenic spacers/NGS/RAD seq?
Try looking into commonly used microsatellite markers in your group (population genetics studies).
Hi, Biswajt, which species do you tested? It may happens that only 1 or 2 marker can be usefull. Do you have DNA sequiencing of these species? May be detailed investigation of the DNA sequiensing give you ideas about marker.
Best wishes!
It depends upon the plant group you are focusing on. Some markers are best for the delimitation of one group but not for the other. You can use the group specific primers for the identification of the taxon.
Hi, Biswajt.
Use the workflow integrative taxonomy by congruence. It can help you.
Best
Hi Biswajt,
I am using RAD-seq to resolve relationships among closely related groups.
Good Luck,
Lalita
nuclear loci always evolve faster than cpDNA, so see if you have any genomic resources for your species.
Alternatively you can design cross-species amplifiable loci from transcriptomes or EST databases using markerminer (http://www.bioone.org/doi/full/10.3732/apps.1400115). I am using it for a genus in the Brassicaceae with no genomic resources - i designed the markers comparing the transcriptomes of three other species. by designing markers that flank introns so you are increasing your chances of picking up differences. Drop me a message if you want more info.
Hello Biswajit:
interesting question, partly because none of the answers posted thus far have addressed the possibility that you are dealing with a single lineage. Maybe the fact that you have used 5 loci and none of these markers have shown any differentiation, is an important fact that is apparently not being acknowledged! In other words, perhaps rather than simply continue your quest for faster evolving markers, which suggests that you are coming into this study with a strong, unshakable preconception that there ARE two species, and you are simply trying to confirm with molecular markers a conclusion that you have already made based on other data (morphology, ecology, etc.). You should consider that it only take a few loci to control certain morphological features. Genomic approach will be the best way to find these "needles in the haystack", but in the meantime, it sounds to me like you have made an important discovery, i.e. that these two lineages are very closely related indeed and by some species definitions (i.e. "phylogenetic species concept") are as single species!
Dear Lalita Simpson,
I am also thinking for doing RAD seq.....could you kindly send me any protocol regarding RAD.....I have read some papers, but, those are purely theoretical...
Hi Biswajit,
RADseq is quite common now and several people are using it. There are companies that do it (check out Cornell's GBS service). $30 per sample in multiples of 95. If you want to do it yourself there will be some setup cost associated with the NGS library prep, even if you choose to make your own adapters instead of buying Illumina ones. I would suggest trying something more simple first such as microsatellites or sequencing a bunch of nuclear loci. You could do one or two transcriptome sequences on a lane of Miseq to develop the markers.
Hello Mark,
What you have suggested earlier, I did not get.. I want to learn the technique. I have read your paper..I really want to do...
Which microsat loci will be useful? I have tried 5-6 loci already....I am trying 2 more hyper variable loci......but...I am not sure about about the result. I am in my last phase of my phd...could you pls help me Mark....
I think cornell is far away from here I work.....what is the cost of RAD if I outsource my samples?
Hi Mark,
I want to design primer using transcriptomes study and further approach to differentiate those closely related species..could you kindly help me or teach me how to do that...
Thanks again..:)
Hi Biswajit,
As long as you can send your samples to Cornell they will run them. It cost me £90 to Fedex mine on dry ice from the UK. If youve read about RASDsreq then youll see that the prep is quite complicated and possibly not something that will work first time. You'll need equipment such as a Pippinprep, magnetic beads and magnetic tube/plate, plus the kits for library prep and adapters are expensive.
I cant recommend microsats to you as I dont know what youre working with. What species/genus is it? Are there genomic resources available? Could you carry out some transcriptome sequencing? In my first post I recommended Markerminer, did you have a look at it?
Yes Mark, I want to do transcriptome sequencing...But, how to do that.
I am working on the genus Nardostachys and Valeriana from family Caprifoliaceae
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