DNA fingerprinting and genetic relationships among willow (Salix spp.)
Ngantcha, Alain Claude
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Given that morphological identification of willow is difficult, willow lines being investigated for their suitability for use as short rotation crops for biomass production in Saskatchewan were investigated with various molecular techniques as possible tools for DNA fingerprinting. Flow cytometry was used to assess variation in nuclear DNA content and thus ploidy level of the lines of the five species (Salix purpurea, Salix eriocephala, Salix sachalinensis, and Salix dasyclados) and three hybrids (S. purpurea x S. miyabeana, S. sachalinensis x S. miyabeana, S. viminalis x S. miyabeana). The DNA content varied between 1.14 and 3.00pg. Ploidy levels of the species varied from triploid to hexaploid while all hybrids were tetraploid. RAPD and ISSR marker systems were used to assess genetic and taxonomic relationships among all willow lines. Of 90 RAPD primers tested, 60 were selected and 99 polymorphic bands scored. Of 35 ISSR primers tested, 19 were selected and 35 polymorphic bands scored. Both RAPD and ISSR dendrograms clustered together lines belonging to the same species and same hybrid combination. A combination of strong and reproducible RAPD and ISSR bands was used to develop identification keys for lines belonging to the same species. The ribosomal RNA gene region, including the entire 5.8S RNA gene and the internal transcribed spacers (ITS1 and ITS2) was amplified and sequenced to assess sequence homology between the five species. The total length of the amplified region was 601bp, with the ITS1, 5.8 S and ITS2 being 223, 163, and 215bp respectively. Intra- and inter-species SNPs were observed, 6 within ITS1, and 3 within ITS2. No polymorphisms were found in the 5.8S gene. The low rate of variation within the sequenced ITS fragment between species supports the monophyly of the five species involved in this study, and confirms their belonging to the subgenus Caprisalix. SCAR primers were designed from species-specific polymorphic nucleotides and applied to the willow collection to test their use for species identification. A species identification key based on SNPs is proposed.
DegreeMaster of Science (M.Sc.)
SupervisorScoles, Graham J.
CommitteeChen, Gang; Knox, Ron; Warkentin, Tom; Fu, Yong-Bi; Coulman, Bruce