MADS-box transcription factors (TFs) have fundamental roles in regulating floral organ formation and flowering time in flowering plants. In order to understand the function of MIKC-type MADS-box family genes in Cyclocarya paliurus (Batal.) Iljinskaja, we first implemented a genome-wide analysis of MIKC-type MADS-box genes in C. paliurus. Here, the phylogenetic relationships, chromosome location, conserved motif, gene structure, promoter region, and gene expression profile were analyzed. The results showed that 45 MIKC-type MADS-box were divided into 14 subfamilies: BS (3), AGL12 (1), AP3-PI (3), MIKC* (3), AGL15 (3), SVP (5), AGL17 (2), AG (3), TM8 (1), AGL6 (2), SEP (5), AP1-FUL (6), SOC1 (7), and FLC (1). The 43 MIKC-type MADS-box genes were distributed unevenly in 14 chromosomes, but two members were mapped on unanchored scaffolds. Gene structures were varied in the same gene family or subfamily, but conserved motifs shared similar distributions and sequences. The element analysis in promoters’ regions revealed that MIKC-type MADS-box family genes were associated with light, phytohormone, and temperature responsiveness, which may play important roles in floral development and differentiation. The expression profile showed that most MIKC-type MADS-box genes were differentially expressed in six tissues (specifically expressed in floral buds), and the expression patterns were also visibly varied in the same subfamily. CpaF1st24796 and CpaF1st23405, belonging to AP3-PI and SEP subfamilies, exhibited the high expression levels in PA-M and PG-F, respectively, indicating their functions in presenting heterodichogamy. We further verified the MIKC-type MADS-box gene expression levels on the basis of transcriptome and qRT-PCR analysis. This study would provide a theoretical basis for classification, cloning, and regulation of flowering mechanism of MIKC-type MADS-box genes in C. paliurus.

Cyclocarya paliurus is an important medical plant owing to the diverse bioactive compounds in its leaves. However, the heterodichogamy with female and male functions segregation within protandry (PA) or protogyny (PG) may greatly affect seed quality and its plantations for medicinal use. To speculate on the factor playing the dominant role in regulating heterodichogamy in C. paliurus, based on phenotypic observations, our study performed a multi comparison transcriptome analysis on female and male buds (PG and PA types) using RNA-seq. For the female and male bud comparisons, a total of 6753 differentially expressed genes (DEGs) were detected. In addition, functional analysis revealed that these DEGs were significantly enriched in floral development, hormone, and GA-related pathways. As the dominant hormones responsible for floral differentiation and development, gibberellins (GAs) in floral buds from PG and PA types were quantified using HPLC-MS. Among the tested GAs, GA3 positively regulated the physiological differentiation (S0) and germination (S2) of floral buds. The dynamic changes of GA3 content and floral morphological features were consistent with the expression levels of GA-related genes. Divergences of GA3 contents at S0 triggered the asynchronism of physiological differentiation between male and female buds of intramorphs (PA-M vs. PA-F and PG-F vs. PG-M). A significant difference in GA3 content enlarged this asynchronism at S2. Thus, we speculate that GA3 plays the dominant role in the formation of heterodichogamy in C. paliurus. Meanwhile, the expression patterns of GA-related DEGs, including CPS, KO, GA20ox, GA2OX, GID1, and DELLA genes, which play central roles in regulating flower development, coincided with heterodichogamous characteristics. These results support our speculations well, which should be further confirmed.

Cyclocarya paliurus is a relict plant species that survived the last glacial period and shows a population expansion recently. Its leaves have been traditionally used to treat obesity and diabetes with the well-known active ingredient cyclocaric acid B. Here, we presented three C. paliurus genomes from two diploids with different flower morphs and one haplotype-resolved tetraploid assembly. Comparative genomic analysis revealed two rounds of recent whole-genome duplication events and identified 691 genes with dosage effects that likely contribute to adaptive evolution through enhanced photosynthesis and increased accumulation of triterpenoids. Resequencing analysis of 45 C. paliurus individuals uncovered two bottlenecks, consistent with the known events of environmental changes, and many selectively swept genes involved in critical biological functions, including plant defense and secondary metabolite biosynthesis. We also proposed the biosynthesis pathway of cyclocaric acid B based on multi-omics data and identified key genes, in particular gibberellin-related genes, associated with the heterodichogamy in C. paliurus species. Our study sheds light on evolutionary history of C. paliurus and provides genomic resources to study the medicinal herbs.

Cyclocarya paliurus is a relict plant species that survived the last Glacial period and shows a population expansion recently. Its leaves have been traditionally used to treat obesity and diabetes with the well-known active ingredient cyclocaric acid B. Here, we present three C. paliurus genomes from two diploids with different flower morphs and one haplotype-resolved tetraploid assembly. Comparative genomic analysis revealed two rounds of recent whole-genome duplication events and identified 691 genes with dosage-effect that likely contribute to adaptive evolution through enhanced photosynthesis and increased accumulation of triterpenoids. Re-sequencing analysis of 45 accessions uncovered two bottlenecks, consistent with the known events of environmental changes, and many selectively swept genes involved in critical biological functions, including plant defense and secondary metabolism biosynthesis. We also proposed the biosynthesis pathway of cyclocaric acid B based on multi-omics data and identified key genes, in particular gibberellin related genes, associated with heterodichogamy in the species. Our research sheds light on evolutionary history and provides genomics resources to study the medicinal herb. External links: https://drive.google.com/file/d/1YDqAk_jvoX_ilb7kSxqs56QfbEiHj9Nm/view?usp=sharing https://pan.baidu.com/s/1XoKEQAjYw0GFgDYvdRT7aw, extraction number: bm0u