I am working on ITS of populus with two universal primers (ITS1 and ITS4 primers), but i some individual two bands amplified.What is the reason of amplification of two band for ITS?
What type of gel are you seeing this pattern on (agarose or PAGE)? If agarose, can you give an indication of the DNA fragment size by comparing against a DNA ladder? Bands in the
"The nuclear rDNA physical maps of Populus nigra, P. deltoides, P. trichocarpa, P. maximowiczii, and P. alba have been built up for the restriction enzymes EcoRI, EcoRV, BamHI, PstI, and SacI. For several clones of each species two to three major unit types coexist in the genome, while several minor units as well as length variant units or new unit types have been found. ... Every species carries several rDNA unit types either different in size (variable length unit) or variable in restriction sites (variable unit types). Four thousands copies of rDNA units were found in P. nigra, P. deltoides, and P. maximowiczii. The clones belonging to the same species carry the same major unit types but are different in their minor variable length units or minor unit types. The hybrid clones carry the sum of the major unit types of the two parental species. These facts suggested the existence of three rDNA loci. ... We conclude that these clones are likely to have a hybridization in their stands but without observable phenotypic consequences."
To judge your amplification specific or not, I suggest that you check your result by analyzing negative control (in PCR reaction with no template). If your PCR amplification is not specific, change the pcr condition to make it sepcific.
The amplification of ITS can be straight forwards nowadays with universal primers. You are mostly facing the issue of the coexistence of paralogs as explained above by Alex.
Both bands can be valid. If your subsequent analyses involve sequencing, then you will have to disentangle the paralogs by cloning or by direct sequencing, both methods coupled with the characterization of features specific to paralogs (GC content or methylation profiles).