Analysis of Differential Mechanisms in Flavonoid Synthesis in the Exocarp of virescens and nigrescens Fruits and Development of KASP Molecular Markers

Background An oil palm. It is an important tropical woody oilseed crop. The fruit contains a relatively high proportion of flavonoids, which are of great nutritional value and have antioxidant, anti - inflammatory effects, and antibacterial functions. The means by which flavonoids are biosynthesized in oil palm is unclear. This study applied LC-MS/MS and RNA-Seq technologies to find out the molecular regulatory mechanism of flavonoids in oil palm exocarp. The exocarps of virescens (FS) and nigrescens (FT) at 95d, 125d, and 185d after pollination were used as experimental materials so as to measure and analyze the metabolome and transcriptome of oil palm exocarps at different developmental stages. The study proposed to find the types of flavonoid metabolites, key synthetic genes, and biological metabolic pathways in oil palm exocarp.Results A total of 274 flavonoids were found in FS, and 275 in FT. In the course of the three developmental stages, the flavonoids in FT were more abundant than in FS. FS had a bigger proportion of flavonoid components, whereas FT contained more anthocyanin components. The differentially expressed metabolites were mainly found in three pathways: flavonoid biosynthesis, flavone and flavonol biosynthesis, and anthocyanin biosynthesis. By analyzing metabolomics and transcriptomics together, the flavonoid biosynthetic pathway was put together, and enzyme genes and metabolites involved in flavonoid synthesis were obtained. In the case of FS, six enzyme genes such as HCT-2, DFR-1, C3'H and seven differential metabolites for example Trifolin, Prunin, Vitexin are involved. For FT, eight enzyme genes such as HCT-1, F3H-1, ANR and nine metabolites, say Isovitexin, Flavosativaside, Baimaside, are involved. The research was able to develop 9 pairs of KASP molecular markers (K2-K10). In the field, they were used to verify how well genotypes and corresponding phenotypes of 30 FS plants and 30 FT plants matched, with an accuracy of 100%. The 9 pairs of these markers are closely linked to the EgDFR gene and can successfully tell FS and FT apart.Conclusions The study gives a theoretical basis for getting clear about the flavonoid synthesis mechanism in the exocarp of oil palm fruits and breeding high - flavonoid oil palm varieties in the future.

背景：油棕（oil palm）是重要的热带木本油料作物。其果实中含有较高比例的黄酮类化合物（flavonoids），具备极高的营养价值，同时具有抗氧化、抗炎及抗菌功能。目前，油棕体内黄酮类化合物的生物合成途径尚不明确。本研究采用液相色谱-串联质谱（LC-MS/MS）与RNA测序（RNA-Seq）技术，解析油棕外果皮中黄酮类化合物的分子调控机制。以授粉后95d、125d及185d的淡绿型油棕外果皮（FS）与深黑型油棕外果皮（FT）为实验材料，对不同发育阶段油棕外果皮的代谢组与转录组进行检测分析，旨在明确油棕外果皮中黄酮类代谢物的种类、关键合成基因及相关生物代谢通路。结果：本研究在FS中共鉴定到274种黄酮类化合物，在FT中共鉴定到275种。在三个发育阶段中，FT的黄酮类化合物总丰度高于FS；FS的黄酮类组分占比更高，而FT则含有更多的花青素类组分。差异表达代谢物主要富集于三条通路：黄酮类化合物生物合成、黄酮与黄酮醇生物合成以及花青素生物合成。通过整合代谢组学与转录组学分析，我们重构了油棕黄酮类化合物的生物合成通路，并筛选得到参与黄酮合成的酶基因与代谢物。其中，FS涉及HCT-2、DFR-1、C3'H等6种酶基因，以及Trifolin、Prunin、Vitexin等7种差异代谢物；FT涉及HCT-1、F3H-1、ANR等8种酶基因，以及Isovitexin、Flavosativaside、Baimaside等9种代谢物。本研究开发了9对KASP分子标记（KASP），并在田间对30株FS材料与30株FT材料的基因型与对应表型的匹配度进行验证，准确率达100%。该9对标记与EgDFR基因紧密连锁，可有效区分FS与FT材料。结论：本研究为阐明油棕果实外果皮中黄酮类化合物的合成机制，以及未来培育高黄酮油棕品种提供了理论依据。