The Sapotaceae family comprises 65–70 genera and over 1250 species, holding significant ecological and economic value. Although previous studies have made some progress in the phylogenetic relationships and classification of Sapotaceae, many issues remain unresolved and require further in-depth research. In this study, we sequenced and assembled the complete chloroplast genomes of 21 plants from 11 genera of Sapotaceae, conducted a comparative genomic analysis, and performed a phylogenetic analysis by incorporating 16 previously published chloroplast genomes of Sapotaceae. The results showed that the chloroplast genome sizes in 21 plants of Sapotaceae range between 157,920 bp and 160,130 bp, exhibiting the typical quadripartite structure. Each genome contains 84–85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes, while the ndhF gene is absent in Pouteria campechiana and Pouteria sapota. The relative synonymous codon usage (RSCU) analysis showed that isoleucine (Ile) is the most commonly used, while the codon for methionine (Met) is the least utilized. Additionally, five highly variable regions (petA-psbJ, psbI-trnS-GGA, rpl2_2-psbA, rps19-rpl2_2, and ycf4-cemA) and two coding sequences, ycf1 and matK, were identified as candidate molecular markers for species differentiation and a phylogenetic analysis within the Sapotaceae family. Phylogenetic trees were reconstructed using complete chloroplast genome sequences and analyzed using ML and BI methods, which revealed that the Sapotaceae family is divided into three distinct clades, each receiving strong statistical support (BS = 100, PP = 1). The intergeneric analysis revealed that Madhuca and Palaquium are sister groups (BS = 91, PP = 1), as are Gambeya and Chrysophyllum (BS = 91, PP = 1). Pouteria and Chrysophyllum are among the larger groups in the Sapotaceae family but the traditional classification boundaries of these genera are unstable and unfeasible, as the current genus boundaries fail to support their natural evolutionary relationships. In the phylogenetic tree, Eberhardtia aurata is placed on a separate branch. The morphological classification system shows that E. aurata has rust-colored pubescence on its branches, abaxial leaf surfaces, petioles, and other areas, which clearly distinguishes it from other genera. This study provides valuable insights into advancing phylogenetic research, population genetics, molecular breeding, and conservation strategies by comparing chloroplast genome structures and characteristics and constructing phylogenetic trees.

Background: RT-qPCR is a powerful strategy for recognizing the most appropriate reference genes, which can successfully minimize experimental mistakes through accurate normalization. Ludisia discolor, recognized for its ornamental value, features little, distinctive blossoms with twisted lips and gynostemium showing chiral asymmetry, together with striking blood-red fallen leaves periodically marked with golden blood vessels. Methods and Results: To ensure the accuracy of qRT-PCR, selecting appropriate reference genes for quantifying target gene expression levels is essential. This study aims to identify stable reference genes during the development of L. discolor. In this study, the entire floral buds, including the lips and gynostemium from different development stages, were taken as materials. Based upon the transcriptome information of L. discolor, nine housekeeping genes, ACT, HIS, EF1-α1, EF1-α2, PP2A, UBQ1, UBQ2, UBQ3, and TUB, were selected in this research study as prospect interior referral genes. The expression of these nine genes were found by RT-qPCR and afterwards comprehensively examined by four software options: geNorm, NormFinder, BestKeeper, and ΔCt. The outcomes of the analysis showed that ACT was the most steady gene, which could be the most effective inner referral gene for the expression evaluation of flower advancement in L. discolor. Conclusions: The results of this study will contribute to the molecular biology research of flower development in L. discolor and closely related species.