Background: Lipid droplets (LDs) present in land plants serve as an essential energy and carbon reserve for seed germination and seedling development. Oleosins, the most abundant structural proteins of LDs, comprise a small family involved in LD formation, stabilization and degradation. Despite their importance, our knowledge on oleosins is still poor in Euphorbiaceae, a large plant family that contains several important oil-bearing species. Results: To uncover lineage-specific evolution of oleosin genes in Euphorbiaceae, in this study, we performed a genome-wide identification and comprehensive comparison of the oleosin family in Euphorbiaceae species with available genome sequences, i.e. castor bean (Ricinus communis), physic nut (Jatropha curcas), tung tree (Vernicia fordii), Mercurialis annua, cassava (Manihot esculenta) and rubber tree (Hevea brasiliensis), and a number of five, five, five, five, eight and eight members were found, respectively. Synteny analysis revealed one-to-one collinear relationship of oleosin genes between the former four (i.e. castor bean, physic nut, tung tree and M. annua) as well as latter two species (i.e. cassava and rubber tree), whereas one-to-one and one-to-two collinear relationships were observed between physic nut and cassava, reflecting the occurrence of one recent whole-genome duplication (WGD) in the last common ancestor of cassava and rubber tree. The presence of five ortholog groups representing three previously defined clades (i.e. U, SL and SH) dates back at least to the Malpighiales ancestor, because they are also conserved in poplar (Populus trichocarpa), a tree having experienced one Salicaceae-specific recent WGD. As observed in poplar, WGD was shown to be the main driver for the family expansion in both cassava and rubber tree. Nevertheless, same retention patterns of WGD-derived duplicates observed in cassava and rubber tree are somewhat different from that of poplar, though certain homologous fragments are still present in rubber tree. Further transcriptional profiling revealed an apparent seed-predominant expression pattern of oleosin genes in physic nut, castor bean and rubber tree. Moreover, structure and expression divergence of paralogous pairs were also observed in both cassava and rubber tree. Conclusion: Comparative genomics analysis of oleosin genes reported in this study improved our knowledge on lineage-specific family evolution in Euphorbiaceae, which also provides valuable information for further functional analysis and utilization of key members and their promoters.

Oleosins (OLEs) are a class of small but abundant structural proteins that play essential roles in the formation and stabilization of lipid droplets (LDs) in seeds of oil crops. Despite the proposal of five oleosin clades (i.e., U, SL, SH, T, and M) in angiosperms, their evolution in eudicots has not been well-established. In this study, we employed Brassicales, an economically important order of flowering plants possessing the lineage-specific T clade, as an example to address this issue. Three to 10 members were identified from 10 species representing eight plant families, which include Caricaceae, Moringaceae, Akaniaceae, Capparaceae, and Cleomaceae. Evolutionary and reciprocal best hit-based homologous analyses assigned 98 oleosin genes into six clades (i.e., U, SL, SH, M, N, and T) and nine orthogroups (i.e., U1, U2, SL, SH1, SH2, SH3, M, N, and T). The newly identified N clade represents an ancient group that has already appeared in the basal angiosperm Amborella trichopoda, which are constitutively expressed in the tree fruit crop Carica papaya, including pulp and seeds of the fruit. Moreover, similar to Clade N, the previously defined M clade is actually not Lauraceae-specific but an ancient and widely distributed group that diverged before the radiation of angiosperm. Compared with A. trichopoda, lineage-specific expansion of the family in Brassicales was largely contributed by recent whole-genome duplications (WGDs) as well as the ancient γ event shared by all core eudicots. In contrast to the flower-preferential expression of Clade T, transcript profiling revealed an apparent seed/embryo/endosperm-predominant expression pattern of most oleosin genes in Arabidopsis thaliana and C. papaya. Moreover, the structure and expression divergence of paralogous pairs was frequently observed, and a good example is the lineage-specific gain of an intron. These findings provide insights into lineage-specific family evolution in Brassicales, which facilitates further functional studies in nonmodel plants such as C. papaya.

In polymerase chain reaction (PCR)-based nucleic acid quantification, the DNA template type, primer/probe sequence, and instrument platform such as real-time quantitative PCR (qPCR) and digital PCR (dPCR) affect the accuracy and reliability of quantitative results. In this study, a plasmid DNA (pDNA) pBI121-screening, genetically modified (GM) rice SDrice genomic DNA (gDNA), and GM rapeseed SDrape gDNA, all carrying the same 11 screening elements, were used to prepare samples of different levels of gDNA and pDNA in a non-GM gDNA background. The comparison of the dPCR assays targeting the 11 screening elements revealed that the primer/probe set is a key factor that affects the accuracy of dPCR quantification. The optimal PCR method for the 11 screening elements was screened out from among the validated qPCR methods. The accuracy of the qPCR quantification of the low-level pDNA and gDNA test samples was low when pDNA was used as a calibrator, whereas that of the dPCR quantification was high and not affected by variations in template type and detection target. The validated dPCR assays targeting one or two elements can be randomly selected to characterize multiple-target pDNA reference materials (RMs). Low-level pDNA RMs with certified values can be used as quality controls for dPCR assays to avoid significant bias in gDNA quantification.