RFLP will be difficult. any thing apart from RFLP?

We use mostly SSR and AFLP. Each has many advantages. The first (SSR) is simple but we have to know the sequences of the primers (microsatellite markers). Therefore, one can start with the second method (AFLP assay) for the species in case of unknown primers sequences.

AFLP

This method does not require knowledge about species-specific marker sequences. The standard procedures of AFLP described by (Vos et al.,1995). High quality genomic DNA (0.5 µg) will be digested with a pair of restriction enzymes (EcoRI and MseI or VspI) as rare and frequent cutter enzymes, respectively, then ligated to double standard (PstI and MseI) adapters. Ligates can be amplified with non-selective primers and following selective amplification by primers extended by three bases. The products will be separated by capillary electrophoresis at an ABI 310 or ABI 3130 sequencer, including a commercially available standard. Peaks representing AFLP markers will scored first automatically with the program GenMapper 5.0 to decrease errors (falsh reads), bin setting may be adjusted manually.

SSR:

Whereas AFLP can be used without prior knowledge about the target species, it has a number of disadvantages, most prominently the anonymous nature of the respective markers. To differentiate allele, SSR is the method of choice, since it allows to estimate directly the degree of heterozygosity for the markers. However we will be in need to derive the primer sequences for DNA sections with microsatellite motifs if the species is located in an isolated position or if we haven't any data about the primers sequences. we only have a low chance of cross-amplification from other species with well known microsatellite primers. A possible alternative way is the use of degenerate primers.

Once established, a SSR assay is much simpler to use than AFLP, as it needs a single PCR step only. All DNA amplifications can be performed on a thermal cycler under the following conditions: 5 min at 94oC, followed by 1 minute at 94oC, 60 s at 55oC and 60S at 72oC for 35 cycles and 7 min at 72oC for final extension. An aliquot of 10 μl of the PCR product is routinely taken for gel electrophoresis to determine if amplification is successful. When the primers detected an amplicon length polymorphism, the samples will be readily scored.

A successful establishment of SSR primers would save both time and money.

SSR you can try on agarose if you have big difference in length. try CAPS.

I think retrotransposons based Molecular markers is very efficient Molecular markers in this regard. There are many PCR based techniques for retrotransposons markers. You can use those markers for studying Biodiversity, phylogenetics, Molecular evolution,.......

I have attached my latest review article about that. You can find many other papers in my publication list that explain and uses such markers in different studies. I hope this will help you.

Article Retrotransposon-based molecular markers for assessment of ge...

SSR can be resolved on metaphopr agarose gels. Latter gives quite good resolution. We have found metaphor agarose gels as a good alternative to PAGE for SSR.

Further, i don't understand why you are needing some more markers systems. What is your exact requirement?