Best way to assess genetic diversity is to use RAPD markers, since it covers whole genome without any specificity and allow to apply in a plant species where primary information is not available. For closely related species, single gene difference may be there as they are developed/originated from same parents. Go for SNP analysis for that. Cytoplasmic male sterility and (nuclear) genetic male sterility specific markers are there in different crops (e.g. Maize T, C and S specific markers are available).

PCR-RFLP can be used for preliminary grouping of your accessions, but if you want to publish the data, it is better to use sequencing of the same regions. It will be a little more expensive comparing to detailed PCR-RFLP with many enzymes.  

Dear Biswajit,

This would be appropriate If you have an idea on diagnostic sites that can delineate within and among species genetic variation. In this case few restriction enzymes with known diagnostic sites would be efficient and allow saving time and money. However, if you want to get accurate results, it is always better to proceed with sequencing as Alex mentioned.

Be very wary of a measure of diversity that is based entirely on a single type of lab marker.  What the conservationists and breeders should really be concerned with is functional diversity, which often can only be clearly and reliably expressed in real-life environments, in 'common-garden' experiments.  While there is typically a broad agreement between marker diversity and functional diversity there can be exceptions which depend on the taxon, the functional traits involved and the marker type.

Choice of marker type can depend on several factors, including: available equipment, cost trends in marker types, and (if known) how well different marker types reflect functional diversity in the particular taxon/taxa.

you'd better to choose markers that are stable, cheap, easy ,simple, available for your diversity analysis according to your real purposes.

Biswajit,

I would summarize all ideas:

1)   Use of  SCoT & intron and matK-RFLP will give you a phylogeny of SCoT &  matK only, not the true taxonomy of your plants. You should use several different methods of different taxonomic power. If the cost of research is critical,  you can try arbitrary PCR markers: ISSR, ITE-PCR, RGA-PCR, AFLP together with locus-specific markers: SSR, STS, PCR-RFLP, but only DNA sequencing for  previously studied fragments will help you to incorporate your plants into known taxonomic trees.  Value of other methods will solely depend on use of good reference material from other studies (if any has been done before for your species).

Dear Alex,

Thank you for your kind suggestions. Actually, if I want to check genetic diversity using matK-RFLP+SCoT of a single plant species, will it be ok to for detecting inter and intra population genetic variability of that plant species and for publication purpose how many restriction enzymes for PCR-RFLP?

Dear Alex,

Kindly could you suggest any highly variable markers for discrimination for closely related species/lower taxonomic level of plant species. 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?