Red-flowered oil-tea camellia (ROC) is an important woody oil species growing in the south, and its oil has high nutritional value. There are four main species of ROC in China, namely, Camellia chekiangoleosa (CCH), Camellia polyodonta (CPO), Camellia semiserrata (CSE) and Camellia reticulata (CRE). Reports on the comprehensive comparative analysis of ROC are limited. This study investigated the fruit characteristics and nutritional components of four ROC fruits, and the results showed that ROC had high oil content with levels of 39.13%–58.84%, especially the CCH fruit, which reached 53.6–58.84%. The contents of lipid concomitants of ROC oil were also substantial, including β-amyrin (0.87 mg/g–1.41 mg/g), squalene (0.43 mg/g–0.69 mg/g), β-sitosterin (0.47 mg/g–0.63 mg/g) and α-tocopherol (177.52 μg/g–352.27 μg/g). Moreover, the transverse diameter(TD)/longitudinal diameter (LD) of fruits showed a significant positive correlation with the oil content, and ROC fruits with thinner peels seemed to have better oil quality, which is similar to the result of the oil quality evaluation obtained by the gray correlation coefficient evaluation method. Four ROC oils were evaluated using the gray correlation coefficient method based on 11 indicators related to the nutritional value of ROC. CCH oil had the highest score of 0.8365, and YS-2 (a clone of CCH) was further evaluated as the best CCH oil. Finally, the results of heatmap analysis showed that triglycerides could be used as a characteristic substance to distinguish CCH oil from the other three ROC oils. The PLSDA (Partial least squares regression analysis) model and VIP (Variable important in projection) values further showed that P/S/O, P/O/O, P/L/L, P/L/Ln, S/S/O, S/O/O and P/S/S (these all represent abbreviations for fatty acids) could be used as characteristic differential triglycerides among the four ROC oils. This study provides a convenient way for planters to assess the nutritional quality of seed oil depending on fruit morphology and a potential way to distinguish between various ROC oils.

MADS-box genes encode transcription factors that affect plant growth and development. Camellia chekiangoleosa is an oil tree species with ornamental value, but there have been few molecular biological studies on the developmental regulation of this species. To explore their possible role in C. chekiangoleosa and lay a foundation for subsequent research, 89 MADS-box genes were identified across the whole genome of C. chekiangoleosa for the first time. These genes were present on all the chromosomes and were found to have expanded by tandem duplication and fragment duplication. Based on the results of a phylogenetic analysis, the 89 MADS-box genes could be divided into either type I (38) or type II (51). Both the number and proportion of the type II genes were significantly greater than those of Camellia sinensis and Arabidopsis thaliana, indicating that C. chekiangoleosa type II genes experienced a higher duplication rate or a lower loss rate. The results of both a sequence alignment and a conserved motif analysis suggest that the type II genes are more conserved, meaning that they may have originated and differentiated earlier than the type I genes did. At the same time, the presence of extra-long amino acid sequences may be an important feature of C. chekiangoleosa. Gene structure analysis revealed the number of introns of MADS-box genes: twenty-one type I genes had no introns, and 13 type I genes contained only 1~2 introns. The type II genes have far more introns and longer introns than the type I genes do. Some MIKC^C genes have super large introns (≥15 kb), which are rare in other species. The super large introns of these MIKC^C genes may indicate richer gene expression. Moreover, the results of a qPCR expression analysis of the roots, flowers, leaves and seeds of C. chekiangoleosa showed that the MADS-box genes were expressed in all those tissues. Overall, compared with that of the type I genes, the expression of the type II genes was significantly higher. The CchMADS31 and CchMADS58 genes (type II) were highly expressed specifically in the flowers, which may in turn regulate the size of the flower meristem and petals. CchMADS55 was expressed specifically in the seeds, which might affect seed development. This study provides additional information for the functional characterization of the MADS-box gene family and lays an important foundation for in-depth study of related genes, such as those involved in the development of the reproductive organs of C. chekiangoleosa.

In order to protect and utilize the germplasm resource better, it is highly necessary to carry out a study on the genetic diversity of Camellia chekiangoleosa Hu. However, systematic research on population genetics analysis of the species is comparatively rare. Herein, 16 highly variable simple sequence repeat (SSR) markers were used for genetic structure assessment in 12 natural C. chekiangoleosa populations. The genetic diversity of C. chekiangoleosa was low (h = 0.596), within which, central populations (such as Damaoshan (DMS), Sanqingshan (SQS), and Gutianshan (GTS)) at the junction of four main mountain ranges presented high diversity and represented the center of the C. chekiangoleosa diversity distribution; the Hengshan (HS) population in the west showed the lowest diversity, and the diversity of the eastern and coastal populations was intermediate. C. chekiangoleosa exhibited a high level of genetic differentiation, and the variation among populations accounted for approximately 24% of the total variation. The major reasons for this situation are the small population scale and bottleneck effects in some populations (HS and Lingshan (LS)), coupled with inbreeding within the population and low gene flow among populations (Nm = 0.796). To scientifically protect the genetic diversity of C. chekiangoleosa, in situ conservation measures should be implemented for high-diversity populations, while low-diversity populations should be restored by reintroduction.

Camellia chekiangoleosa Hu. (C. chekiangoleosa) is a typical high-altitude oil-tea Camellia species. Due to altitude being an important factor affecting crop growth and quality, the influence of environmental conditions associated with low (60 m) and high (600 m) altitudes on the economic and quality characteristics of fruit ripening was assessed in this study. Our investigations showed that altitude has no influence on the growth pattern of C. chekiangoleosa fruit shells and seed oils, and the differences in samples between different altitudes gradually decreased with the ripening of C. chekiangoleosa. Nevertheless, mature C. chekiangoleosa fruit shells and seed oils from low and high altitudes showed some differences. Specifically, the fruit shells of C. chekiangoleosa cultivated in low-altitude areas contained more soluble sugar, protein, total polyphenols, total flavonoids, and tea saponin. Meanwhile, low-altitude cultivation elevated the abundance of α-tocopherol, β-sitosterol, β-amyrinol, flavonoids, and polyphenols in mature seed oils but decreased the oil yield. Moreover, few effects of altitude on fatty acid composition were observed in mature seed oils. Cluster and receiver operating characteristic (ROC) analysis indicated that the influence of altitude on the quality of mature seed oils was strongly associated with oil yield and α-tocopherol. Taken together, the present study suggests that when cultivating C. chekiangoleosa in low-altitude regions, more energy should be devoted to improving oil yield. The results of the fruiting process and quality trait variation in C. chekiangoleosa during the low-altitude introduction process can provide an important theoretical basis for the introduction and cultivation of this oil-tea species.

Color variation in plant leaves has a significant impact on their photosynthesis and plant growth. Camellia chekiangoleosa yellow-leaf mutants are ideal materials for studying the mechanisms of pigment synthesis and photosynthesis, but their mechanism of leaf variation is not clear. We systematically elucidated the intrinsic causes of leaf yellowing in the new Camellia chekiangoleosa variety ‘Diecui Liuji’ in terms of changes in its cell structure, pigment content, and transcript levels. This study indicates that the incomplete structure of chloroplast-like vesicles, the decrease in blue-green chlorophyll a, and the increase in yellow-green chlorophyll b in yellowing leaves are the direct causes of yellowing-leaf formation. The high expression of genes that catalyze the degradation of chlorophyll a (PAO and RCCR) and its conversion to chlorophyll b (CAO) in yellowing leaves leads to a decrease in the chlorophyll a content, while the low expression of CLH genes is the main reason for the increase in the chlorophyll b content. We also found transcription factors such as ERF, E2F, WRKY, MYB, TPC, TGA, and NFYC may regulate their expression. RT-qPCR assays of 12 DEGs confirm the RNA-seq results. This study will provide a foundation for investigating the transcriptional and regulatory mechanisms of leaf color changes.

The WRKY gene family is essential for controlling a variety of plant physiological functions, yet the involvement of specific WRKY members in pigment biosynthesis and accumulation in Camellia chekiangoleosa remains unexplored, particularly in anthocyanins and carotenoids, which play crucial roles in the pigmentation of C. chekiangoleosa. This study systematically identified 87 WRKY genes across 15 chromosomes in C. chekiangoleosa through bioinformatic approaches. Further structural and phylogenetic analyses of these TFs enabled their classification into six different subgroups. WRKY family expansion was shown to be mostly driven by tandem duplication. W-box elements, which can be binding sites for WRKY transcription factors, were present in a number of biosynthetic genes in the pigment production pathway. Yeast one-hybrid assay confirmed that five WRKY transcription factors (CchWRKY15/24/33/47/76) directly bind to the promoter regions of two key biosynthetic genes, CchPSY1 and Cch4CL1. Intriguingly, among the five WRKYs tested, the expression levels of CchWRKY15, CchWRKY33, and CchWRKY47 showed the strongest positive associations with flavonoid accumulation (p < 0.05, Pearson correlation analysis).These findings provide novel insights into the evolutionary patterns, transcriptional regulation, and functional characteristics of CchWRKYs, while elucidating their possible regulatory mechanisms in the fruit coloration of C. chekiangoleosa.