Yellowhorn (Xanthoceras sorbifolium Bunge) is an economically important tree species in China. Its seeds are distinguished by exceptionally high levels of both oil and protein, making it an attractive model for studying the coordination of storage reserve accumulation. To elucidate the molecular mechanisms underlying these two traits, we conducted the first comprehensive multi-omics study, integrating proteomic and transcriptomic analyses during kernel development. Untargeted proteomic analysis identified 4861 proteins, of which 820 exhibited differential expression across different kernel developmental stages. Transcriptome analysis identified 18,935 differentially expressed genes (DEGs) across developmental stages, and functional annotation further identified 66 DEGs associated with lipid biosynthesis and 39 DEGs related to storage protein synthesis. Interestingly, the integrated analysis revealed 22 transcription factors (TFs) from the bZIP, NF-Y, MYB, and C3H families, potentially playing pleiotropic regulatory roles in lipid and protein metabolism. LEC1 and bZIP38 exhibited peak expression at the S2 stage, temporally synchronized with the critical phase of seed oil/protein biosynthesis. Our multi-omics data demonstrate that the synergy between oil and protein biosynthesis in Yellowhorn is likely orchestrated by a shared transcriptional network, rather than resulting from resource competition as commonly observed in other oil crops. This study reveals conserved transcriptional networks coordinating lipid-protein co-allocation in seeds, providing molecular insights for engineering high-oil/protein cultivars through precision breeding.

Summary statement This study identified a novel miR5021‐CoWRKY28 module that regulates anthocyanin content through targeting CoCHS3 , thus improving anthracnose resistance. These findings provide a key clue between the miRNA‐transcription factor module and anthocyanin biosynthesis pathway for plant disease resistance.

Background: Eastern Tongren City, Guizhou Province, China, possesses abundant wild germplasm resources of Camellia oleifera Abel.; however, there is a lack of systematic evaluation, and the promotion of superior varieties is insufficient. This study aimed to evaluate 21 trait indices of 189 wild C. oleifera accessions from four regions in the Tongren area to clarify their variation characteristics, assess the effects of altitude on trait expression, and identify candidate germplasms with outstanding comprehensive performance. Methods: A total of 21 traits spanning fruit morphology, oil content, fatty acid composition, and bioactive components (tocopherols, squalene, and polyphenols) were measured. Principal Component Analysis (PCA) was employed to construct a comprehensive evaluation score (Zn) for quantitative ranking and screening. Trait differences between a low-altitude group (400–800 m, n = 139) and a high-altitude group (800–1200 m, n = 50) were compared using Welch’s t-test. Results: The germplasms exhibited abundant phenotypic variation, with coefficients of variation (CV) ranging from 5.29% (total unsaturated fatty acids) to 124.42% (beta + gamma-tocopherol). Bioactive components showed the highest variability, while fatty acid composition was relatively stable. Altitude had a significant effect on six of the 21 traits. Seed oil content and kernel oil content were significantly higher in the high-altitude group, with mean differences of 5.92 and 5.74 percentage points, respectively (both p < 0.001). However, oleic acid, total unsaturated fatty acids, fruit morphological traits, and most bioactive components showed no significant altitudinal differences (p > 0.05). The first five principal components explained 65.0% of the total variance. CL40 achieved the highest comprehensive score (Zn = 4.15), followed by MJX2 (Zn = 2.56). Among the top 10 individuals, eight were from the low-altitude group. Conclusions: This study revealed rich phenotypic variations and distinct altitudinal effects among wild C. oleifera germplasms in eastern Guizhou. The superior germplasms identified (such as CL40 and MJX2) can serve as candidate materials for locally adapted variety improvement. This study was primarily based on single-season phenotypic data, and the genetic stability of the selected germplasms should be validated through clonal trials and molecular marker analysis in future research.

Yellowhorn (Xanthoceras sorbifolium Bunge.) is an important woody oilseed species but suffers from low seed yield due to imbalanced female-male flower ratios. Through key hormones and genes potentially regulating flower sex differentiation were identified, such as auxin (IAA) related genes (SAUR50, ARF4, ARF5, PIN1C, and LAX5) and strigolactone (SL) related genes (SPL9, D14, and SMXL7) were found to be associated with the promotion of female flower differentiation, while abscisic acid (ABA) related genes (PHI-1, NCED1, and CYP707A2) and jasmonic acid (JA) related genes (TIFY10B, TIFY5A, and TIFY9) were implicated in male flower differentiation. A total of 10 miRNA-mRNA regulatory modules related to flower sex differentiation were revealed. Notably, the SL related miR156-SPLs and IAA related miR167-ARF8 were identified to be involved in female flower differentiation, whereas the ABA related miR159-MYB101 module was associated with male flower differentiation. Additionally, 12 lncRNA-mRNA regulatory modules were discovered, including MSTRG.44661.2-ANT/SPL9, MSTRG.7848.2-ARF8, MSTRG.47452.3-FAR2, and MSTRG.18934.1-QKY for promoting female flower differentiation, and MSTRG.36342.2-CALS5, MSTRG.30760.1-GLOX1/PRK1/PRK4, MSTRG.25556.1-MYB101, MSTRG.10529.1-MIK2, and MSTRG.47537.2-AGL104 for promoting male flower differentiation. Furthermore, two competing endogenous RNA (ceRNA) networks, such as MSTRG.41858.1-miR156-SPL9, were identified to promote female flower differentiation. The expression patterns of 7 miRNAs, 8 lncRNAs, and their target genes were validated using qRT-PCR. The interactions within the MSTRG.41858.1-miR156-SPL9 ceRNA network were validated through dual-luciferase reporter assays and RNA pulldown experiments. This study establishes ncRNA-mediated regulation of sex differentiation as a novel molecular strategy for optimizing flower sex ratios in yellowhorn, a critical factor for enhancing oil yield in this woody oilseed crop. The identified ceRNA network provides a direct target for genetic engineering to increase female flower proportion. Furthermore, the hormone-ceRNA crosstalk mechanism revealed here offers a paradigm for sex determination research in other oil-producing plants, accelerating the development of sex-controlled breeding systems in perennial crops.

Sea buckthorn (Hippophae rhamnoides L.) is a perennial woody oil and horticultural crop from the Elaeagnaceae family with high nutritional and economic value. The novel cultivar 'Gaoyou No.1' (PVR No. 20190345) exhibits superior seed oil yield and quality. However, the lack of a high-quality reference genome for this cultivar has greatly hindered molecular studies on the accumulation of unsaturated fatty acids (FAs) and triacylglycerols (TAGs) in its seed oil. Here, we report a chromosome-level genome assembly of 'Gaoyou No.1', with a final size of 1.27 Gb and a scaffold N50 of 99.72 Mb, alongside 26 429 annotated protein-coding genes. Repetitive sequences accounted for 72.35% of the genome. Comparative genomic analysis dated the divergence of H. rhamnoides from Juglans regia to approximately 107.5 million years ago. We identified 14 298 structural variations (SVs) in 'Gaoyou No.1' compared with a common cultivated sea buckthorn accession. Through whole-transcriptome profiling, 49 and 55 enzymatic genes involved in the FA and TAG biosynthesis pathways were identified, respectively; 21 of these genes harbored SVs, in which the copy number variations of SAD_0.613 and LPCAT_ 16.398 enhanced their expression via a gene dosage effect, and further promoted the oil biosynthesis in 'Gaoyou No.1'. Furthermore, 90 lncRNA–mRNA pairs, 88 miRNA–mRNA pairs, and 42 lncRNA–miRNA–mRNA regulatory modules were predicted. The interactions within 12 key modules, such as lncRNA051245–miRn43–FAD2 and lncRNA111000–miRn275–LPAT were experimentally verified. We propose that these SVs and regulatory modules collectively contribute to the improved seed oil yield and quality of 'Gaoyou No.1', providing promising targets for genetic improvement. Our research yields novel insights into the molecular mechanisms underlying high FA and TAG accumulation in sea buckthorn, while also furnishing valuable genomic resources for evolutionary research in Elaeagnaceae and molecular breeding development.

1IntroductionSea buckthorn (Hippophae rhamnoides L.) is a woody oil tree known for its fruits, which are a rich source of bioactive compounds, including carotenoids and flavonoids (Ciesarova et al., 2020; Mihal et al., 2023). In addition, the unique fatty acid composition of the fruit pulp oil, especially the high content of omega-7 monounsaturated palmitoleic acid, which is rare in plants, contributes to the nutritional benefits of its products (Sola Marsinach and Cuenca, 2019). In this regard, sea buckthorn products are used in medicine, cosmetics, and nutraceuticals (Gatlan and Gutt, 2021; Guo et al., 2022; Zuchowski, 2023). In addition to cultivation for fruit production, sea buckthorn is also used for ecological restoration due to its high resistance to extreme conditions (Ruan et al., 2013).Sea buckthorn is mainly cultivated in China (2.07 million ha), India (0.02 million ha), Romania (0.02 million ha), Mongolia (0.02 million ha), Russia (0.01 million ha), and Pakistan (0.01 million ha) (Nybom et al., 2023). Thus, 90% of sea buckthorn resources are located in China (Singh, 2022). However, the pioneer in sea buckthorn breeding was Russia, where selection of H. rhamnoides ssp. mongolica Rousi started in 1933 and allowed the development of a wide range of high-yield varieties with high-quality fruits (Singh and Zubarev, 2014). In contrast, breeding of sea buckthorn in China started later, mainly with H. rhamnoides ssp. sinensis Rousi (Nybom et al., 2023). Varieties of H. rhamnoides ssp. mongolica are characterized by large fruits, high yield, high oil content, and lower acidity compared to H. rhamnoides ssp. sinensis varieties, which are better adapted to abiotic and biotic stressors (Nybom et al., 2023). Sea buckthorn breeding does not stand still, new improved varieties are being developed and genetic data can contribute to this. However, only a few DNA markers potentially useful for sea buckthorn breeding are known. Markers were proposed to distinguish Hippophae species and subspecies, including H. rhamnoides ssp. sinensis and H. rhamnoides ssp. mongolica (Liu et al., 2015; Liu et al., 2016; Liu et al., 2018; Piao et al., 2022). Hippophae species are dioecious, and attempts were made to develop DNA markers to identify sex, but these markers do not always work in genetically diverse material (Korekar et al., 2012; Das et al., 2017; Zhou et al., 2018; Zeng et al., 2024a; Zeng et al., 2024b). Markers associated with oil content in fruits (Ding et al., 2016) and genes involved in flavonoid biosynthesis (Zhang et al., 2024) were identified. Several works were performed to search for genes associated with resistance of Hippophae species to biotic and abiotic stressors (Nybom et al., 2023). In recent years, high-quality genome assemblies of H. rhamnoides (with sizes of 849, 730, and 919 Mb) (Wu et al., 2022; Yu et al., 2022; Yang et al., 2024), Hippophae tibetana (957 and 1453 Mb) (Wang et al., 2022b; Zhang et al., 2024), and Hippophae gyantsensis (716 Mb) (Chen et al., 2024) were obtained. However, a very limited number of sea buckthorn genotypes were studied using whole-genome sequencing. Whole-genome sequencing of only 40 wild H. rhamnoides ssp. mongolica and H. rhamnoides ssp. sinensis representatives and 15 cultivated H. rhamnoides ssp. mongolica varieties was performed by Chinese researchers (Yu et al., 2022). Therefore, there is a lack of genomic data for varieties of sea buckthorn. The aim of the present study was to fill this gap by performing whole-genome sequencing of the unique set of 55 varieties of Russian breeding, which are likely to be significantly different from the Chinese varieties and characterized by valuable traits. These data can significantly expand the knowledge of the diversity of H. rhamnoides at the whole-genome level and provide the necessary data for the development of genetic technologies for sea buckthorn breeding.2Materials and Methods2.1Plant MaterialTo cover the diversity of sea buckthorn cultivated in Russia, a set of 56 accessions representing 55 varieties of H. rhamnoides L. was formed: one replicate for 54 varieties (one tree for each variety) and two biological replicates for the variety Elizaveta (two different trees). The following valuable characteristics were considered: weight, flavor, shape, and color of the fruits and differences in origin (Table 1). Characteristics of sea buckthorn varieties were assessed according to Kondrashov et al. (Kondrashov et al., 1999). Dormant shoots of the selected genotypes were collected at the Federal Altai Scientific Center of Agrobiotechnologies (Barnaul, Russia) in April 2023. The shoots were placed in containers with water in a room with a temperature of ~22 °C for one week. When the leaves appeared, they were collected in tubes, frozen in liquid nitrogen, and stored in a low-temperature freezer until DNA extraction.2.2DNA ExtractionDNA was extracted using the Magen HiPure Plant DNA Mini Kit (Magen, Guangzhou, China). The quality and quantity of DNA were evaluated using NanoDrop 2000C (Thermo Fisher Scientific, Waltham, MA, USA), Qubit 4.0 (Thermo Fisher Scientific), and agarose gel electrophoresis (2% agarose).2.3Whole-Genome SequencingThe QIAseq FX DNA Library UDI Kit (Qiagen, Chatsworth, CA, USA) was used for DNA library preparation. Quantity and quality of DNA libraries were assessed using Qubit 4.0 (Thermo Fisher Scientific) and Qsep1-Plus (Bi-Optic, New Taipei City, Taiwan). Genome sequencing was performed on a NovaSeq 6000 (Illumina, San Diego, CA, USA) with a read length of 150 + 150 bp.2.4Sequencing Data AnalysisThe obtained Illumina reads were processed with Trimmomatic 0.39 (TRAILING:28, SLIDINGWINDOW:4:17, MINLEN:40) (Bolger et al., 2014). The processed reads were mapped to the annotated H. rhamnoides genome from the CNGB Nucleotide Sequence Archive (https://db.cngb.org/cnsa), project ID CNP0001846 (Wu et al., 2022), and VAF (Variant Allele Frequencies) values were calculated for genome regions corresponding to genes (exons and introns) using PPLine (Krasnov et al., 2015). Genetic distances between sea buckthorn varieties were calculated and clustered with Ward's method (ward.D2) in PPLine (Krasnov et al., 2015).3Preliminary Data AnalysisA representative set of 56 accessions comprising 55 sea buckthorn varieties (for the variety Elizaveta, two different trees were analyzed) was formed from the unique collection of the Federal Altai Scientific Center of Agrobiotechnologies (Barnaul, Russia). The selected varieties had different fruit characteristics and different origins in order to maximize the diversity of the analyzed set (Table 1).Whole-genome sequencing was performed and at least 23 Gbases of raw Illumina data were obtained for each accession, which corresponded to more than 25× genome coverage (raw Illumina reads were deposited to NCBI SRA, BioProject PRJNA1177110). After mapping the reads to the annotated H. rhamnoides reference genome, data on about 4 million DNA polymorphisms in genes were obtained (lists of DNA polymorphisms were deposited to Zenodo, https://zenodo.org/records/13999625). These data are useful for studying the diversity of allelic variants for specific genes, especially those that may be associated with valuable traits, such as the content of bioactive compounds and other fruit characteristics and resistance to stressors. It is worth noting that a significant part of the identified DNA polymorphisms was present in all analyzed sea buckthorn varieties, indicating that they are genetically distinct from the used reference genome. In addition, genetic distances between the accessions were calculated to evaluate their relationships (Supplementary Table 1).To visualize the relationships of the studied varieties based on DNA polymorphisms in gene sequences, a dendrogram was constructed (Figure 1). Cluster I was the most distinct and included KP-686 (Kyrgyz ecotype), Dunayskaya, and Yantarnaya Yagoda, which are not varieties of Altai breeding and probably have significant differences at the genome level from the other studied accessions. The same cluster included 175-02-01, obtained by crossing varieties of Altai breeding, and its position in the dendrogram is not expected and requires additional research.The remaining sea buckthorn varieties were divided into four clusters. Cluster II included Afina and all studied progenies of this variety, namely 625-08-01, 625-14-1, 762-14-1, and 763-14-1. It can be assumed that Afina and sea buckthorn genotypes obtained with its participation are genetically quite different from the other studied varieties of Altai breeding. In addition, 111-05-01, 258-03-01, and 42-68-2, which are believed to be unrelated to Afina, were in Cluster II, which is difficult to explain from a genealogical point of view.Cluster III clearly distinguished a group of sea buckthorn varieties with Panteleevskaya in their lineages. Thus, this group is likely to be significantly different from other studied sea buckthorn genotypes at the genome level.Cluster IV included 14 varieties, among which the genetic relationships were not as clear as in the first three clusters, but they were still present. Thus, a group of four Novosibirsk accessions was isolated: Triumf, Zarnitsa, 681-09-01, and 708-13-1, with Triumf being the parental form for 681-09-01 and 708-13-1. Ulala and its progeny 125-02-01 were also in this cluster. Two varieties with Panteleevskaya in their lineages were also in Cluster IV: 22-02-2003 and 226-00-01. In general, however, this cluster contained a mixture of quite different sea buckthorn varieties.Cluster V contained 23 accessions. In this cluster, as in other clusters, some relationships corresponding to lineages were observed. For example, Rosinka and Sudarushka, which entered this cluster, have common roots. In addition, varieties Essel and 218-03-06 have the genotype 89-72-6a in their lineages. 89-72-6a is very interesting in terms of strong inheritance of large fruit size. In this respect, it is the progenitor of many varieties, most of which were present in Cluster V. The exception was the variety Aureliya, which was in Cluster IV. Other relationships can also be traced in Cluster V. For example, Lyubimaya clone is a seedless mutant of the variety Lyubimaya. Several closely related groups were also isolated: Elizaveta (two biological replicates) and its progeny 2016-00-1, Chechek and its progeny Ognivo, Chuyskaya and its progeny Klavdiya and 111-10-2, and Panteleevskaya and its progeny 114-13-1.In general, the dendrogram obtained by us on the basis of DNA polymorphisms in all sea buckthorn genes annotated in the used reference genome (Wu et al., 2022) reflected well the known data on the relationship of the studied genotypes. The research on H. rhamnoides performed by Yu et al. using whole-genome sequencing allowed the authors to separate wild genotypes of H. rhamnoides ssp. mongolica from cultivated ones, as well as to separate H. rhamnoides ssp. sinensis accessions into a separate group (Yu et al., 2022). However, we were unable to find any other work that characterized representative sets of sea buckthorn genotypes using whole-genome sequencing (NCBI PubMed, https://pubmed.ncbi.nlm.nih.gov/; Google Scholar, https://scholar.google.com; accessed October 28, 2024). Meanwhile, whole-genome sequencing and linkage mapping is an urgent need for sea buckthorn studies (Sharma, 2022).Data on the diversity of sea buckthorn varieties at the genomic level are of great value in understanding the extent to which selection has affected the gene pool of this crop and what patterns can be traced by analyzing the genetic data. We studied the sea buckthorn varieties of Russian breeding, which has a long history. The forms with valuable traits created by Russian breeders became the progenitors of many varieties all over the world (Singh, 2022), so the obtained by us data are of special value. In addition, the evaluation of genetic relationships of different accessions is important for breeders when selecting parental forms for crosses.Recently, there has been an increasing number of articles devoted to the beneficial properties of sea buckthorn (Wang et al., 2022a; Chen et al., 2023; Mihal et al., 2023; Nybom et al., 2023; Teng et al., 2024; Xu et al., 2024), but in terms of genetics, this crop is still relatively understudied (Sharma, 2022). Indeed, several high-quality genome assemblies of H. rhamnoides were obtained (Wu et al., 2022; Yu et al., 2022; Yang et al., 2024) and some transcriptome studies were performed (Bansal et al., 2018; Ye et al., 2018; Gao et al., 2022; Lyu et al., 2022; Yu et al., 2022). A number of works were also devoted to fatty acid synthesis in sea buckthorn and genes/microRNAs involved in this process (Ding et al., 2018; Ding et al., 2019; Ding et al., 2022; Yu et al., 2022; Arkhipov et al., 2024). However, the genetic determinants and their diversity remain unknown for most of the key traits that define the value of sea buckthorn varieties, including carotenoid content, fruit shape and flavor. In this context, data on DNA polymorphisms in gene sequences obtained for a representative set of accessions characterized by phenotype will allow the search for associations between allelic variants of genes and valuable traits. These data are the basis for the development of marker-assisted and genomic selection of sea buckthorn, which are increasingly used in breeding practice for other agricultural plants (Xu et al., 2020; Hasan et al., 2021; Thudi et al., 2021; Dmitriev et al., 2022; Werner et al., 2023; Mangal et al., 2024).4ConclusionsH. rhamnoides is a valuable crop whose fruits are rich in bioactive compounds with health benefits. To date, there is a lack of genetic data for varieties of sea buckthorn. This fact hinders the identification of genetic determinants of valuable traits and limits the efficiency of breeding. In the present study, we analyzed a representative set of 55 valuable H. rhamnoides varieties of Russian breeding with different fruit characteristics and diverse lineages. Whole-genome sequencing was performed on the Illumina platform, and at least 25× genome coverage was obtained for each accession. Based on the sequencing data, DNA polymorphisms were identified in genomic regions corresponding to genes. These polymorphisms were used to evaluate the genetic relationships of the studied sea buckthorn varieties. We revealed genetically distinct groups of accessions that mostly corresponded to the lineages of the genotypes. Our data are important for assessing the effect of selection on sea buckthorn diversity and for evaluating the genetic relationship of different varieties, which is useful for breeders when selecting parental forms for crosses. The obtained data on genomic sequences of 55 H. rhamnoides varieties in combination with information on valuable traits of their fruits are the basis for identification of quantitative trait loci (QTL) and quantitative trait nucleotides (QTN) for further development of DNA tests. This will be the basis for marker-assisted selection of sea buckthorn. The obtained information on DNA polymorphisms is also necessary to study the diversity of genes, including those that may determine valuable traits, such as fruit characteristics. This will help to promote genomic breeding of H. rhamnoides. Thus, our data can benefit both basic and applied research on sea buckthorn.

Sea buckthorn is a vital woody oil species valued for its role in soil conservation and its bioactive seed oil, which is rich in unsaturated fatty acids and other compounds. However, low seed oil content and small seed size are the main bottlenecks restricting the development and utilization of sea buckthorn. In this study, we tested the seed oil content and seed size of 12 sea buckthorn cultivars and identified the key genes and transcription factors involved in seed development and lipid biosynthesis via the integration of UID RNA-seq (Unique Identifiers, UID), WGCNA (weighted gene co-expression network analysis) and qRT-PCR (quantitative real-time PCR) analysis. The results revealed five cultivars (CY02, CY11, CY201309, CY18, CY21) with significantly higher oil contents and five cultivars (CY10, CY201309, CY18, CY21, CY27) with significantly heavier seeds. A total of 10,873 genes were significantly differentially expressed between the S1 and S2 seed developmental stages of the 12 cultivars. WGCNA was used to identify five modules related to seed oil content and seed weight/size, and 417 candidate genes were screened from these modules. Among them, multiple hub genes and transcription factors were identified; for instance, ATP synthase, ATP synthase subunit D and Acyl carrier protein 1 were related to seed development; plastid–lipid-associated protein, acyltransferase-like protein, and glycerol-3-phosphate 2-O-acyltransferase 6 were involved in lipid biosynthesis; and transcription factors DOF1.2, BHLH137 and ERF4 were associated with seed enlargement and development. These findings provide crucial insights into the genetic regulation of seed traits in sea buckthorn, offering targets for future breeding efforts aimed at improving oil yield and quality.

Sea buckthorn (Hippophae rhamnoides L.) is an oil crop with health benefits. Its fruits are rich in unsaturated fatty acids (FAs); however, the FA composition of the seeds and pulp differs significantly. To evaluate the expression levels of gene families that play a major role in FA biosynthesis, the transcriptomes of seeds and pulp at four fruit development stages were sequenced for five sea buckthorn varieties with diverse characteristics: Elizaveta, Inya, KP-686, Panteleevskaya, and Triumf. The results revealed that FAD3 (07426) and FAD3 (05528) are likely key genes for linolenic acid synthesis in seeds, while FAD2 (21624) is likely the main contributor to linoleic acid synthesis in both seeds and pulp. SAD (18830) primarily contributes to oleic acid synthesis in seeds, while SAD (18830) and SAD (26748) contribute to its synthesis in pulp. FATA (14745) and FATA (14109) are also implicated in FA synthesis in sea buckthorn fruits. Changes in the content of the main FAs in seeds and pulp correlated with the expression levels of the corresponding genes. KP-686 and Triumf differed the most from other varieties. These results are important for analyzing tissue-specific gene expression in seeds and pulp of sea buckthorn fruits, and they are promising for developing sea buckthorn varieties with improved oil composition.

KAS II (β-ketoacyl-acyl carrier protein (ACP) synthases II), FAT (fatty acid thioesterases), SAD (stearoyl-ACP desaturase), and FAD (fatty acid desaturases) are the vital gene families involved in fatty acid (FA) synthesis in Hippophae rhamnoides L. However, information on the number and location of these genes and which ones are key to the formation of FAs in fruit seeds and pulp was not complete. Our study aimed to solve this issue using the available genomic sequences and transcriptome data that we obtained. We compared the protein sequences of sea buckthorn with those of Arabidopsis thaliana and checked for the presence of conserved domains. As a result of structure and phylogenetic analyses, 4 KAS II, 8 FAT, 9 SAD, and 12 FAD genes were identified in the H. rhamnoides genome, which were classified into subfamilies: KAS II, FATA, FATB, FAD2, FAD3, FAD6, and FAD7/8. To analyze the expression of the identified genes, we sequenced the transcriptomes of sea buckthorn seeds and fruit pulp at four development stages, as well as leaves. The analysis revealed representatives of the FAT, SAD, and FAD families with high tissue-and stage-specific expression in seeds and pulp. These genes are likely to play a key role in the biosynthesis of sea buckthorn FAs. The obtained results may help to establish the precise biosynthesis mechanisms of FAs and will promote the breeding of new sea buckthorn varieties that have oil with a defined FA composition.

ScopeCerebral ischemia‐reperfusion (IR) injury stands as a prominent global contributor to disability and mortality. Nervonic acid (NA), a bioactive elongated monounsaturated fatty acid, holds pivotal significance in human physiological well‐being. This research aims to explore the prophylactic effects and fundamental mechanisms of NA in a rat model of cerebral IR injury.Methods and resultsThrough the induction of middle cerebral artery occlusion, this study establishes a rat model of cerebral IR injury and comprehensively assesses the pharmacodynamic impacts of NA pretreatment. This evaluation involves behavioral analyses, histopathological examinations, and quantification of serum markers. Detailed mechanisms of nervonic acid's prophylactic effects are revealed through fecal metabolomics and 16S rRNA sequencing analyses. Our findings robustly support nervonic acid's capacity to ameliorate neurological impairments in rats afflicted with cerebral IR injury. Beyond its neurological benefits, NA demonstrates its potential by rectifying metabolic perturbations across diverse pathways, particularly those pertinent to unsaturated fatty acid metabolism. Additionally, NA emerges as a modulator of gut microbiota composition, notably by selectively enhancing vital genera likeLactobacillus.ConclusionThese comprehensive findings highlight the potential of incorporating NA as a functional component in dietary interventions aimed at targeting cerebral IR injury.

Hippophae rhamnoides is a valuable crop whose fruits are rich in bioactive compounds with health benefits. To date, there is a lack of genetic data for varieties of sea buckthorn. This fact hinders the identification of genetic determinants of valuable traits and limits the efficiency of breeding. In the present study, we analyzed a representative set of 55 valuable H. rhamnoides varieties of Russian breeding with different fruit characteristics and diverse lineages. Whole-genome sequencing was performed on the Illumina platform and at least 25x genome coverage was obtained for each accession. Based on the sequencing data, DNA polymorphisms were identified in genome regions corresponding to genes. These polymorphisms were used to evaluate the genetic relationships of the studied sea buckthorn varieties. We revealed genetically distinct groups of accessions that mostly corresponded to the lineages of the genotypes. Our data are important for assessing the effect of selection on sea buckthorn diversity and for evaluating the genetic relationship of different varieties, which is useful for breeders when selecting parental forms for crosses. The obtained information on DNA polymorphisms is also necessary to study the diversity of genes, including those that may determine valuable sea buckthorn traits, including fruit characteristics. Thus, our data can benefit both basic and applied research on sea buckthorn. ### Competing Interest Statement The authors have declared no competing interest.

为考察不同产地文冠果种仁黄酮类成分的差异,以山东潍坊的60份和辽宁大连的58份文冠果种仁为原料,采用LC-MS/MS法检测其黄酮类成分及含量,并对两地文冠果种仁黄酮类成分含量的差异进行了对比分析.结果表明:118份文冠果种仁中均检测出5种黄酮类成分,其中芦丁含量为1.422～40.704 μg/g,表没食子儿茶素含量为2.513～6.406 μg/g,没食子儿茶素没食子酸酯含量为1.994～4.574 μg/g,二氢槲皮素含量为1.102～4.452 μg/g,没食子儿茶素含量为0.806～4.057 μg/g;两地文冠果种仁黄酮类成分含量大小依次为芦丁＞表没食子儿茶素＞没食子儿茶素没食子酸酯＞二氢槲皮素＞没食子儿茶素;山东潍坊不同样树种仁中黄酮类成分总含量介于10.518～25.445 μg/g之间,样树WF927的黄酮类成分总含量最高;辽宁大连不同样树种仁中黄酮类成分的总含量介于8.347～52.087 μg/g之间,样树DL392的黄酮类成分总含量最高;两地文冠果种仁黄酮类成分含量间差异显著,其中辽宁大连文冠果种仁中黄酮类成分总含量平均值(19.491 μg/g)极显著高于山东潍坊的(16.320 μg/g);两地118份文冠果种仁中,黄酮类成分总含量最高的为 DL392(52.087 μg/g)、DL395(43.711 μg/g)和 DL347(40.481 μg/g).

为了提高沙棘种子的品质,选取大连民族大学校园内的沙棘'实优1号'的种子为研究对象,用野生型拟南芥(Col-0)作为 目的基因的转化受体,以沙棘种子油脂合成关键基因GPD1和DGAT1为载体,通过In-fu-sion 连接技术构建HrGPD1和HrDGAT1基因双价表达载体pCGD,采用农杆菌蘸花法获取转基因拟南芥植株,利用甲酯化和氯仿甲醇法对和野生型的拟南芥植株种子进行脂肪酸不同组份的比例及含油率检测.结果表明:拟南芥转基因2代与野生型相比,拟南芥2代种子油酸、亚油酸的比例分别提高了 46.70％和9.35％,但种子含油率未明显提高.因此,同时表达沙棘HrGPD1和HrDGAT1基因提高了油酸、亚油酸含量,为高油酸沙棘优良品种的培育和改良提供了基因材料.

Yellowhorn (Xanthoceras sorbifolium) seeds can have as high as 67% oil content and are especially rich in oleic acid, linoleic acid, and nervonic acid. Exploration of the lipid biosynthesis regulatory network is essential for increasing the yellowhorn oil content. Long non-coding RNAs (lncRNAs) play important roles in various plant biological processes; however, there is no report on the identification of lncRNAs involved in yellowhorn seed development and lipid biosynthesis affecting oil production. We performed whole transcriptome sequencing of yellowhorn seeds at four developmental stages and identified 16,920 putative lncRNAs. Among them, 325 lncRNAs were revealed to trans-regulate 58 key genes in fatty acid (FA) and triacylglycerol (TAG) biosynthesis pathways. Of these, ECR-2–LNC_009778 was found to be involved in nervonic acid biosynthesis and DGAT-1–LNC_009778 was beneficial to TAG accumulation. sRNA-seq was performed, and 55 microRNAs (miRNAs) were found to target 26 genes involved in FA and TAG biosynthesis; miR396a-4 targets FAD2, affecting linoleic acid biosynthesis, and miR156f-5p targets PDAT-2, contributing to TAG accumulation. Interestingly, 30 lncRNA–miRNA–gene modules involved in FA and TAG biosynthesis were identified, in which the KCS11-1–miR156g-2–LNC_000849 module was found to participate in nervonic acid synthesis, and the DGAT-2–miR172j–LNC_005874 module was assumed to contribute to the accumulation of TAG. Our results constitute the first comprehensive identification of lncRNAs in developing seeds of yellowhorn and serve as a new theoretical reference for improving oil content in the future.

[目的]为深入挖掘辽西地区沙棘种质资源,筛选出果实品质好的沙棘优良单株,为沙棘野外选种及果实加工利用提供依据.[方法]以辽西地区18个优良沙棘单株为原材料,对其果实的百果质量、横径、纵径、可溶性糖含量、维生素E含量等 14 个主要性状及成分进行测定,并采用相关性分析及主成分分析方法对其果实品质进行综合评价.[结果]18个优株的百果质量为11.38～26.99 g,横径长为6.21～8.35 mm,纵径长为5.46～7.91 mm,两径均值为5.84～7.85 mm,果柄长度为1.39～2.24 mm,果糖、葡萄糖和可溶性糖含量分别为4.07～21.27、1.04～13.70和36.82～78.65 mg·g-1 FW,维生素E含量为42.43～145.61 μg·g-1 FW,可溶性固形物含量为 8.83%～18.23%FW,总酚含量为 5.07～10.74 mg·g-1 FW,可滴定酸含量为 1.65%～3.63%FW,类胡萝卜素含量为 0.08～0.36 mg·g-1 FW,总黄酮含量为 1.59～11.07 mg·g-1 FW.沙棘果实主要性状及成分有明显相关性,经主成分分析将 14 个品质性状综合成 4 个主成分因子,其累积贡献率可达 81.514%,第 1 主成分(果形因子)的特征值为 4.919,方差贡献率为 35.138%;第 2 主成分(甜度因子)的特征值为 2.755,方差贡献率为19.679%;第3主成分(营养因子)的特征值为2.149,方差贡献率为15.349%;第4主成分(活性因子)的特征值为 1.589,方差贡献率为 11.346%.[结论]经综合评价筛选出 6 个综合得分高的沙棘优株,即JP17、JP15、JP18、JP16、JP14 和JP10,可通过深入研究作为辽西地区推广种植的中国沙棘优良品种.

Camellia oleifera Abel is a highly valued woody edible oil tree, which is endemic to China. It has great economic value because C. oleifera seed oil contains a high proportion of polyunsaturated fatty acids. C. oleifera anthracnose caused by Colletotrichum fructicola, poses a serious threat to C. oleifera growth and yield and causes the benefit of the C. oleifera industry to suffer directly. The WRKY transcription factor family members have been widely characterized as vital regulators in plant response to pathogen infection. Until now, the number, type and biological function of C. oleifera WRKY genes are remains unknown. Here, we identified 90 C. oleifera WRKY members, which were distributed across 15 chromosomes. C. oleifera WRKY gene expansion was mainly attributed to segmental duplication. We performed transcriptomic analyses to verify the expression patterns of CoWRKYs between anthracnose-resistant and -susceptible cultivars of C. oleifera. These results demonstrated that multiple candidate CoWRKYs can be induced by anthracnose and provide useful clues for their functional studies. CoWRKY78, an anthracnose-induced WRKY gene, was isolated from C. oleifera. It was significantly down-regulated in anthracnose-resistant cultivars. Overexpression of CoWRKY78 in tobacco markedly reduced resistance to anthracnose than WT plants, as evidenced by more cell death, higher malonaldehyde content and reactive oxygen species (ROS), but lower activities of superoxide dismutase (SOD), peroxidase (POD), as well as phenylalanine ammonia-lyase (PAL). Furthermore, the expression of multiple stress-related genes, which are associated with ROS-homeostasis (NtSOD and NtPOD), pathogen challenge (NtPAL), and pathogen defense (NtPR1, NtNPR1, and NtPDF1.2) were altered in the CoWRKY78-overexpressing plants. These findings increase our understanding of the CoWRKY genes and lay the foundation for the exploration of anthracnose resistance mechanisms and expedite the breeding of anthracnose-resistant C. oleifera cultivars.

ObjectiveSea buckthorn was one of important woody oil A low for soil and water conservation and sand prevention and control, and its seed oil contained rich bioactive components. A low seed oil content was the main bottleneck restricting the development and utilization of sea buckthorn. The objective of this study was to clone key genes of GPD1 (glycerol-3-phosphate dehydrogenase), DGAT1 (diacylglycerol acyltransferase 1) and DGAT2 (diacylglycerol acyltransferase 2), which were related to seed oil biosynthesis in sea buckthorn; and test their function by heterologous over expression. This will provide a scientific basis for breeding sea buckthorn cultivars with high seed oil.MethodAccording to the transcriptome sequencing results of sea buckthorn, HrGPD1, HrDGAT1 and HrDGAT2 genes were cloned using the cDNA template of seabuckthorn seeds. Pcambia1300-mCherry expression vector was constructed by in fusion ligation technology. The expression vector was transferred into GV3101 by the Agrobacterium-mediated method, and the Arabidopsis thaliana strains with an overexpressed of the three key genes were obtained by Agrobacterium impregnation. The expressions of three target genes in transgenic A. thaliana were determined by qRT-PCR. The oil contents of T2 transgenic and wild-type A. thaliana seeds were detected by chloroform methanol method.ResultThe correct gene sequences of HrGPD1, HrDGAT1 and HrDGAT2 were amplified by PCR, HrGPD1 consisted of 975 nucleotides encoding a protein of 324 amino acids with a calculated molecular mass of 35.55 kDa and a predicted pI of 5.36. HrDGAT1 consisted of 1 608 nucleotides encoding a protein of 535 amino acids with a calculated molecular mass of 61.24 kDa and a predicted pI of 8.84. HrDGAT2 consisted of 993 nucleotides encoding a protein of 330 amino acids with a calculated molecular mass of 37.21 kDa and a predicted pI of 9.72. GPD1 protein contained one conserved function domain, which had been identified as a glycerol-3-phosphate dehydrogenase family members. HrGPD1, ZiGPD1 and MnGPD1 had been grouped together, suggested that HrGPD1 had a close relationship with GPD1 protein among Ziziphus jujube and Morus notabilis. DGAT1 and DGAT2 proteins contained one conserved domain respectively, which was a member of membrane-bound O-acyltransferase family. DGAT1 had a closer evolutionary relationship with Ziziphus jujube. DGAT2 had a closer evolutionary relationship with Malus domestica and Pyrus x bretschneideri. The A. thaliana strains with stably over expressed HrGPD1, HrDGAT1 and HrDGAT2 genes were obtained by Agrobacterium transformation. The oil contents in T2 generation seeds of transgenic A. thaliana plants with overexpressed HrGPD1, HrDGAT1 and HrDGAT2 genes increased by 5.09%, 4.73% and 2.51%, respectively, compared to wild type.ConclusionOverexpressing the total coding region sequence of HrGPD1, HrDGAT1 and HrDGAT2 were cloned and their functions were characterized. The HrGPD1, HrDGAT1 and HrDGAT2 genes of seabuckthorn increased the seed oil content of transgenic A. thaliana strains. These results have provided genetic materials for the breeding of seabuckthorn cultivars with high seed oil contents, which has important significance for increasing the seed oil production of seabuckthorn.

Few flower buds in a high-yield year are the main factors restricting the yield of Camellia oleifera in the next year. However, there are no relevant reports on the regulation mechanism of flower bud formation. In this study, hormones, mRNAs, and miRNAs were tested during flower bud formation in MY3 (“Min Yu 3,” with stable yield in different years) and QY2 (“Qian Yu 2,” with less flower bud formation in a high-yield year) cultivars. The results showed that except for IAA, the hormone contents of GA3, ABA, tZ, JA, and SA in the buds were higher than those in the fruit, and the contents of all hormones in the buds were higher than those in the adjacent tissues. This excluded the effect of hormones produced from the fruit on flower bud formation. The difference in hormones showed that 21–30 April was the critical period for flower bud formation in C. oleifera; the JA content in MY3 was higher than that in QY2, but a lower concentration of GA3 contributed to the formation of the C. oleifera flower bud. JA and GA3 might have different effects on flower bud formation. Comprehensive analysis of the RNA-seq data showed that differentially expressed genes were notably enriched in hormone signal transduction and the circadian system. Flower bud formation in MY3 was induced through the plant hormone receptor TIR1 (transport inhibitor response 1) of the IAA signaling pathway, the miR535-GID1c module of the GA signaling pathway, and the miR395-JAZ module of the JA signaling pathway. In addition, the expression of core clock components GI (GIGANTEA) and CO (CONSTANS) in MY3 increased 2.3-fold and 1.8-fold over that in QY2, respectively, indicating that the circadian system also played a role in promoting flower bud formation in MY3. Finally, the hormone signaling pathway and circadian system transmitted flowering signals to the floral meristem characteristic genes LFY (LEAFY) and AP1 (APETALA 1) via FT (FLOWERING LOCUS T) and SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CO 1) to regulate flower bud formation. These data will provide the basis for understanding the mechanism of flower bud alternate formation and formulating high yield regulation measures for C. oleifera.