Reverse transcription quantitative PCR is a widely used method to detect gene expressions. To obtain accurate expression results, the selection of proper reference genes is important and necessary. However, related works concerning reference gene selection have not been carried on many plant species, especially endangered ones. The aim of the present study was to select dependable reference genes for expression normalization of an endangered plant species with medicinal and ornamental value: Sinocalycanthus chinensis (Calycanthaceae). Nine reference genes with stable expressions were chosen for further analysis according to transcriptomic sequencing data from S. chinensis. The expression stability of these candidate genes in inner and outer petals at different floral developmental stages and in many different tissues was then analyzed with the geNorm and NormFinder software. The reference genes were evaluated by normalizing the expression of the anthocyanidin synthase gene in the outer and inner petals at different floral developmental stages to further verify the expression stability of these genes. Elongation factor 1-alpha (ScEF) and 50S ribosomal protein L27 were found to be the two most stable genes in the overall ranking of all the samples and different tissues. Furthermore, ScEF and protein phosphatase 2A were stably expressed in all petal samples. Moreover, among the nine candidate reference genes, phosphoglycerate kinase performed poorly in all sample sets. Our results will help to obtain reliable expression data in molecular studies of S. chinensis.

ABSTRACT. We investigated the genetic diversity of the southern flounder Paralichthys lethostigma. Microsatellite-enriched libraries were constructed and novel microsatellite markers were developed and applied for genetic detection of wild populations. Cross-species amplification was also conducted in five pleuronectiforme species. Of 45 randomly selected and sequenced clones, 43 contained a CA or GA repeat motif. Fourteen pairs of primers were designed to investigate the polymorphism and genetic structure of a wild population collected from North Carolina State coastal waters. Two loci were monomorphic and 12 loci were polymorphic. The number of alleles per polymorphic locus ranged from 2 to 16, with an average of 7.3, and the expected heterozygosity per locus ranged from 0.10 to 0.92, with an average of 0.58. Crossspecies amplification showed that most of the markers could successfully amplify Paralichthys olivaceus DNAs, few markers amplified in Verasper variegatus and Verasper moseri, and none of them could amplify Scophthatmus maximus and Cynoglossus semilaevis DNAs. The isolated polymorphic markers would be useful for the genetic breeding and assessment of genetic variation within the genus Paralichthys.