Elucidation of myricetin biosynthesis in Morella rubra of the Myricaceae.

Flavonols are health-promoting bioactive compounds important for plant defense and human nutrition. Quercetin (Q) and kaempferol (K) biosynthesis have been studied extensively while little is known about myricetin (M) biosynthesis. The roles of flavonol synthases (FLSs) and flavonoid 3',5'-hydroxylase (F3'5'H) in M biosynthesis in Morella rubra, a member of the Myricaceae rich in M-based flavonols, were investigated. The level of MrFLS transcripts alone did not correlate well with the accumulation of M-based flavonols. However, combined transcript data for MrFLS1 and MrF3'5'H showed a good correlation with the accumulation of M-based flavonols in different tissues of M. rubra. Recombinant MrFLS1 and MrFLS2 proteins showed strong activity with dihydroquercetin (DHQ), dihydrokaempferol (DHK), and dihydromyricetin (DHM) as substrates, while recombinant MrF3'5'H protein preferred converting K to M, amongst a range of substrates. Tobacco (Nicotiana tabacum) overexpressing 35S::MrFLSs produced elevated levels of K-based and Q-based flavonols without affecting M-based flavonol levels, while tobacco overexpressing 35S::MrF3'5'H accumulated significantly higher levels of M-based flavonols. We conclude that M accumulation in M. rubra is affected by gene expression and enzyme specificity of FLS and F3'5'H as well as substrate availability. In the metabolic grid of flavonol biosynthesis, the strong activity of MrF3'5'H with K as substrate additionally promotes metabolic flux towards M in M. rubra.

Genome-wide identification and analysis of the MADS-box gene family and its potential role in fruit development and ripening in red bayberry (Morella rubra).

The MADS-box gene family encodes transcription factors and plays an important role in plant growth and the development of flower and fruit. A perennial dioecious plant, the red bayberry genome has been published recently, providing the opportunity to analyze the MADS-box gene family and its role in fruit development and ripening. Here, we identified 54 MADS-box genes in the red bayberry genome, and classified them into two types based on phylogenetic analysis. Thirteen Type I MADS-box genes were subdivided into three subfamilies and 41 Type II MADS-box genes into 13 subfamilies. A total of 46 MADS-box genes were distributed across eight red bayberry chromosomes, and the other eight genes were located on the unmapped scaffolds. Transcriptome analysis suggested that the expression of most Type II genes was higher than Type I in five female tissues. Moreover, 26 MADS-box genes were expressed during red bayberry fruit development and ten of them showed high expression. qRT-PCR showed that the expression of MrMADS01 (SEP, MIKCC), with differences between the pale pink and red varieties, increased significantly at the final ripening stage, suggesting it may participate in ripening as positive regulator and related to anthocyanin biosynthesis. These results provide some clues for future study of MADS-box genes in red bayberry, especially in ripening process.

Phylogeny of Morella rubra and Its Relatives (Myricaceae) and Genetic Resources of Chinese Bayberry Using RAD Sequencing.

Phylogenetic relationships among Chinese species of Morella (Myricaceae) are unresolved. Here, we use restriction site-associated DNA sequencing (RAD-seq) to identify candidate loci that will help in determining phylogenetic relationships among Morella rubra, M. adenophora, M. nana and M. esculenta. Three methods for inferring phylogeny, maximum parsimony (MP), maximum likelihood (ML) and Bayesian concordance, were applied to data sets including as many as 4253 RAD loci with 8360 parsimony informative variable sites. All three methods significantly favored the topology of (((M. rubra, M. adenophora), M. nana), M. esculenta). Two species from North America (M. cerifera and M. pensylvanica) were placed as sister to the four Chinese species. According to BEAST analysis, we deduced speciation of M. rubra to be at about the Miocene-Pliocene boundary (5.28 Ma). Intraspecific divergence in M. rubra occurred in the late Pliocene (3.39 Ma). From pooled data, we assembled 29378, 21902 and 23552 de novo contigs with an average length of 229, 234 and 234 bp for M. rubra, M. nana and M. esculenta respectively. The contigs were used to investigate functional classification of RAD tags in a BLASTX search. Additionally, we identified 3808 unlinked SNP sites across the four populations of M. rubra and discovered genes associated with fruit ripening and senescence, fruit quality and disease/defense metabolism based on KEGG database.

Molecular phylogeny of Myricaceae: a reexamination of host-symbiont specificity.

The phylogeny of 13 species of Myricaceae, the most ancient actinorhizal family involved in a nitrogen-fixing symbiosis with the actinomycete Frankia, was established by the analysis of their rbcL gene and 18S-26S ITS. The phylogenetic position of those species was then compared to their specificity of association with Frankia in their natural habitat and to their nodulation potential determined on greenhouse-grown seedlings. The results showed that Genus Myrica, including M. gale and M. hartwegii, and Genus Comptonia, including C. peregrina, belong to a phylogenetic cluster distinct from the other Myrica species transferred in a new genus, Morella. This grouping parallels the natural specificity of each cluster with Comptonia-Myrica and Morella being nodulated by two phylogenetically divergent clusters of Frankia strains, the Alnus and Elaeagnaceae-infective strains clusters, respectively. Under laboratory conditions, Comptonia and Morella had a nodulation potential larger than under natural conditions. From this study it appears that the Myricaceae are split into two different specificity groups. It can be hypothesized that the early divergence of the genera led to the selection of genetically diverse Frankia strains which is contradictory to the earlier proposal that evolution has proceeded toward narrower promiscuity within the family.

Characterization of six microsatellite loci in Myrica faya (Myricaceae) and cross amplification in the endangered endemic M. rivas-martinezii in Canary Islands, Spain

Six novel polymorphic microsatellite markers were isolated from enriched libraries in Myrica faya Ait., recently renamed Morella faya, (fayatree, firetree, or firebush) in order to examine the genetic diversity in natural populations. Also, test cross-specific amplification and genetic diversity in Myrica rivas-martinezii, which is endemic on the Canary islands. Microsatellite loci were screened in 225 individuals of both species from different islands of the Canarian archipelago. All markers were successfully amplified from both Myrica species, with an average number of 6.5 and 9.3 alleles per locus in M. rivas-martinezii and M. faya, respectively. There was no evidence for linkage disequilibrium between loci, and the probability of null alleles ranged from 0.01 to 0.17.