Tilia is a complex genus in the family Malvaceae that has high ecological and economical values. Owing to the lack of sufficient distinguishable morphological and molecular characteristics, interspecific relationships in this genus are not clear. Chloroplast (cp) genomes are small, meanwhile most angiosperms usually undergo matrilineal inheritance. Consequently, they can be used in molecular systematics and phylogenetic analyses. Here, we sequenced and assembled cp genomes from T. endochrysea, T. tomentosa, T. miqueliana, T. americana and T. cordata, and compared them with those of seven previously reported Tilia species. Similar gene contents, gene orders and GC contents existed among the 12 cp genomes, which ranged from 162,564 to 162,855 bp and encoded 113 unique genes. Abundant simple sequence repeats (119–127) and dispersed repeats (97–135) were detected in Tilia cp genomes. In total, 11 hypervariable regions were identified that could be suitable for species identification and phylogenetic studies. A phylogenetic analysis of Malvaceae based on 5 hypervariable genes (matK + ndhF + rpoB + rpoC2+ycf1) revealed that all eight subfamilies were monophyletic groups. Additionally, the genus Tilia was divided into three groups on the basis of all 521 molecular variation loci. The current study provides valuable insights into the genomic evolution of the genus Tilia.

Tilia endochrysea Handel-Mazzetti 1926, distributed in the southern margin of Chinese Subtropical Zone, is the only one species in genus Tilia has dehiscent fruit. The whole chloroplast genome of T. endochrysea was 162,838 bp with 36.46% GC content, including a large single-copy (LSC) region of 91,264 bp, a small single-copy (SSC) region of 20,414 bp, and a pair of inverted repeats (IRs) of 51,160 bp. The chloroplast genome encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic tree shows T. endochrysea was the basal taxon in Tilia and closely related to Craigia yunnanensis (Smith et Evans 1921).

Quantitative real-time polymerase chain reaction (qRT-PCR) is a rapid and effective approach toward detecting the expression patterns of target genes. The selection of a stable reference gene under specific test condition is essential for expressing levels of target genes accurately. Tilia miqueliana, considered endangered, is a prominent native ornamental and honey tree in East China. No study has evaluated the optimal endogenous reference gene for qRT-PCR analysis in T. miqueliana systematically. In this study, fifteen commonly used reference genes were selected as candidate genes, and the stabilities of their expressions were assessed using four algorithms (GeNorm, NormFiner, BestKeeper, and DeltaCt) in nine experimental datasets. The final integrated evaluation was performed using a comprehensive analysis algorithm (RefFinder). Finally, a target MYB transcription factor gene (TmMYB) was used to verify the accuracy of the candidate reference genes. The results showed that PP2α was the most stable in tissue set, while RPS13 and SAMCD were optimal for heat and cold stress, respectively. Under waterlogging stress, PP2α and TUB were the most stable genes in the leaves and roots, respectively. EF1α and PP2α were optimal for drying stress in leaf and root tissues. TUB and ACT7 were the most stable genes in the leaf and root tissues under salt stress. This is the first systematic evaluation of candidate reference genes in T. miqueliana, and it will benefit future studies on expression and functional analysis of target genes in T. miqueliana.