Isolation and DNA-Marker Based Genotyping of Individual Pollen Grains

Dry pollen of diploid Solanum clone 9507-04, Cotton chromosomal substitution line and Echinacea purpurea „Magnus‟ were isolated and their cells contents released in individual tubes containing water or pollen germination media, respectively.Six polymorphic markers from individual pollen grains of the diploid potato were amplified using two random amplified polymorphic DNA (RAPD) primers, i.e., UBC291 and UBC504. The procedures for primer extension pre-amplification (PEP) for genome and subsequent amplified fragment length polymorphism (AFLP) analysis with 13 primers pairs were tested on the upland cotton line. The cotton parental AFLP markers were scored for their presence or absences in the 35 pollen samples and majority of the markers were found monomorphic as expected from the gametes of a near isogenic line.PEP and AFLP procedures for linkage mapping were standardized usingpurpleconeflower pollen grains. From 64 possible AFLP primer pairs,37.5% amplified 104 markers for coneflowerthat were linked in 11 linkage groupsranging from 13 to 134 centimorgans (cM). The 409 cM sizedlinkage map constructed forE. purpurea „Magnus‟genome had average distance of 2.4 cM to 22 cMfor the two to 55 polymorphic markers identifiedwithin the 11 groups.The protocols described in this report would serve to enhance breeding efficiencies in plantsimportant for food,fiber, ornamental and medicinal industries.