Polymorphism in Alnus based Frankia of Darjeeling.

DNA samples extracted from the root nodules of Alnus nepalensis, collected from 10 different locations of Darjeeling hills, were used to assess the genetic diversity of Frankia. The DNA samples from the nodules of naturally growing plants were used as templates in PCR, targeting different genomic regions of Frankia, namely distal, middle and proximal parts of 16S rRNA gene and nifH-D IGS region with locus specific primers. The PCR products were digested with a number of frequent (4-base) cutter restriction endonucleases. Bands were scored as present (1) or absent (0) and the clustering was done using NTSYSpc. Distinct polymorphism was found among the nodules collected from different parts of the region and those of same geographic area. These results demonstrate that genetic diversity is indeed present among the naturally occurring Frankia of Darjeeling, India.

Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts.

While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.