Abstract:

Objective: Ostrya multinervis is a species endemic to China, and it is listed as a rare and endangered wild plant, and a species with extremely small population in Zhejiang Province. The wild O. multinervis trees are found to be very rare in Zhejiang Province with very few young-aged plants and a declining population. During the evolution process, the chloroplast genomes are relatively conserved and thus provide useful information for genetic relationship analysis and phylogenetic studies. In the present study, based on the assembly of chloroplast genome, sequence characteristics were determined to confirm its structure and gene organization. By phylogenetic analysis, relationships and taxonomic ranks with its relative were identified. Methods: Genomic DNA of O. multinervis was extracted by using CTAB method, and a library was constructed for high-throughput sequencing with HiSeq X Ten with the paired-end strategy. The chloroplast genome was assembled by using NovoPlasty, and PCR was applied to isolate the border sequences following sequencing verification. SSR loci were detected by MISA and a phylogenetic tree was generated using PhyML 3.0. Results: A total of 19 869 412 clean reads were obtained by getting rid of both adaptors and low quality sequences. The result indicated that the complete O. multinervis chloroplast genome was 159 583 bp in length containing a typical quadripartite structure, and the lengths of large single copy, small single copy, and inverted repeat were 88 514 bp, 18 953 bp, and 26 058 bp, respectively. The O. multinervis chloroplast genome contains 131 genes, with 87 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. Among them, ycf1 and ycf15 were pseudogenes, and their sequences were shorter than normal ones. A total of 58 SSR loci were predicted from O. multinervis plastome, and 53 of which were mononucleotide repeats. Multiple sequence comparison showed the highest similarity between O. multinervis and O. rehderiana (99.1%) and the lowest was between O. multinervis and O. japonica (98.7%). The best fit model for constructing a phylogenetic tree was GTR+G+I, and the Akaike information criterion and Bayesian information criterion were 556 643.629 64 and 556 954.642 07, respectively. The phylogenetic analysis indicated that 12 plants could be divided into two groups, and Alnus japonica and A. rubra from the tribe Betuleae were grouped into one clade, while 10 plants from tribe Coryleae were grouped into another clade. O. multinervis and the other three Ostrya species clustered in the same clade with 100% of bootstrap support. Conclusion: Based on the high-throughput sequencing, the chloroplast genome of O. multinervis was assembled, and its structure and gene organizations were confirmed. Higher similarities in chloroplast genome sequences were found between O. multinervis and other three Ostrya species, and they shared the same clade. The assembly, sequence comparison and phylogenetic analysis provide a basis for genetic structure and population diversity studies in the future.

Key words: Ostrya multinervis, chloroplast genome, assembly, phylogenetic analysis