Abstract: [Objective] Betula halophila was recorded as an endangered species in the list of plant species with extremely small populations (PSESPs).In this study,we used high-throughput sequencing technology to sequence its chloroplast (cp) genome to understand the structure of B.halophila cp genome as well as the potential simple sequence repeats (SSRs) markers.We also compared the cp genome of B.halophila with those of the phylogenetically related species to understand the hotspot region in Betulaceae.[Method] Total genomic DNA was extracted from fresh leaf samples using the Plant Genomic DNA Kit (Tiangen Biotech).The HiSeq Xten platform was used for library construction and sequencing.MITObim v1.8 and NOVOplasty were used to assemble trimmed reads with the cp genome of B.nana as the reference.[Result] Totally,30 000 000 reads were obtained after trimming.The B.halophila cp genome (Accession Number MG674393 in NCBI) was a typical circular double-stranded DNA molecule with 160 648 bp in length,and its quadripartite structure was similar to most angiosperms,which comprised of LSC region (89 553 bp,GC content 33.7%),SSC regions (18 159 bp,GC content 29.7%) and IRs regions (IRa and IRb,26 689 bp each,GC content 42.5%).Four unique rRNAs,31 tRNAs,and 79 protein-coding genes were annotated.We further detected 91 SSRs,of which 33 SSRs were composed of A or T,and 56,13,and 22 SSRs were at the LSC,SSC and IRs,respectively.The sliding window analysis showed three highly variable regions (hotspots) under a criterion of nucleotide variability >0.2:ndhC-trnV,petA-psbJ,and rpl22-rps19.All of these hotspots locate at the LSC region.In contrast,the SSC and IRs regions are relatively conservative.In addition,four genes of ycf1,rpoC1,rpl2,and ndhA were signaled with positive selection.Phylogenomic reconstruction with 13 dicotyledonous plants revealed that nine of the 11 nodes had 100% supports for grouping in both MP and NJ trees.The phylogenomic analysis indicated that B.halophila was close to B.nana and sister with Ostrya rehderiana.[Conclusion] Via the high-throughput sequencing and the bioinformatic analyses of the cp genome of B.halophila,we found the similar organization,gene contents and SSRs distribution with B.nana cp genomes.Three identified highly variable regions in LSC are applicable for DNA barcodes for species identification,plant breeding,and phylogenetic inference.Four positively selected genes deserve further investigation.It provides important genetic information for the future protection of B.halophila.

Key words: Betula halophila, chloroplast genome, high-throughput sequencing, phylogenetic analysis