Abstract:

【Objective】 The phylogenetic relationships among genera in the Salicaceae family have remained controversial, largely because the available genetic markers lack polymorphisms. More efficient and highly variable genetic markers are needed to address lingering questions about taxonomic relationships within the Salicaceae family. Compared with the cytoplasmic genome, single-copy nuclear genes (SCNGs) are inherited bi-parentally, carry a large number of information sites, and are orthologous, and therefore can better reflect phylogenetic relationships among species and satisfy the requirements of different taxa. 【Method】 Based on the 15 SCNG markers of old Salicaceae members (Populus and Salix) and the internal transcribed spacer (ITS) sequences published in previous studies, PCR amplification and sequencing were carried out in 4 species in the Salicaceae family: Scolopia chinensis, Flacourtia ramontchi, Casearia velutina and Homalium cochinchinense. The primers were designed based on the sequencing results. Then, we used the representative species of 6 different genera of Salicaceae (Bennettiodendron leprosipes, Xylosma congesta, Dovyalis hebecarpa, Itoa orientalis, Idesia polycarpa and Poliothyrsis sinensis) to verify the application of the primers. Polymorphism and neutral tests were performed on the new primers using 20 individuals in a population of S. chinensis. Maximum parsimony and Bayesian inference methods were used to construct the phylogenetic trees by combining data (SCNGs+ITS). 【Result】 Six pairs of primers (5 SCNG markers and ITS) with an aligned length ranging from 297 to 716 bp were successfully identified from 10 genera. The results of polymorphism analysis of the 6 primers showed that the number of haplotypes (H) ranged from 3 to 9, the average nucleotide difference (κ) was 0.644-2.278, the nucleotide diversity (π) and the Watterson’s parameter (θ_w) were 0.001 04-0.004 51 and 0.001 19-0.004 75, respectively. Neutral test results indicated that all loci were consistent with neutral evolutionary assumptions. Two phylogenetic trees showed similar topology and both could be classified well with high support values based on combined data. 【Conclusion】 The six markers developed in this study are anticipated to enrich molecular markers for species in the Salicaceae family, and provide useful information for elucidating the phylogenetic relationships and genetic diversity within Salicaceae.

Key words: Salicaceae, single-copy nuclear gene, polymorphism, phylogenetic relationship