Phylogenomics and diversification drivers of the Eastern Asian - Eastern North American disjunct Podophylloideae.

Evolutionary and biogeographic processes determine species richness patterns of vascular plants between Eastern Asia (EA) and Eastern North America (ENA). However, the strikingly higher species richness of EA relative to ENA remains poorly understood from this perspective. Here, we studied the relative importance of biogeographical, evolutionary and ecological factors underlying differences in species richness between EA and ENA in Podophylloideae (Berberidaceae, Ranunculales; in total 10 spp. in EA vs. 2 spp. in ENA). Based on large-scale transcriptome data, our phylogenomic analyses strongly supported Podophylloideae and its two multi-species genera, i.e. Dysosma (EA) and Diphylleia (EA/ENA), as monophyletic groups. Sinopodophyllum hexandrum (EA) was identified as sister to the remainder of Podophylloideae. Dysosma (7 spp.) was recovered as sister to Podophyllum peltatum (ENA), forming an EA-ENA disjunct pair with a strong bias of species diversity in the EA counterpart. Our biogeographic analyses support the 'out-of-Tibet' hypothesis, suggesting that Podophylloideae started to diversify in the Himalaya-Hengduan Mountains (Mid-Miocene) and migrated eastward (since the Late Miocene) into Central-eastern China, Japan, and ENA (only P. peltatum and Diphylleia cymosa). Overall, we conclude that the striking species diversity anomaly between EA and ENA in Podophylloideae may be explained by a combination of (1) a longer period of time available to accumulate species in EA; and (2) a greater diversification rate in EA, which might have been promoted by greater physiographic and environmental heterogeneity in this region.

Genome survey sequencing and genetic diversity of cultivated Akebia trifoliata assessed via phenotypes and SSR markers.

Akebia trifoliata (Lardizabalaceae) is an important medicinal plant with multiple pharmacological effects. However, the lack of genomic information had limited the further excavation and utilization of this plant. An initial survey of the genome A. trifoliata was performed by next-generation sequencing, and then the genome size was inferred by flow cytometry. The whole genome survey of A. trifoliata generated 61.90 Gb of sequence data with approximately 95.51 × coverage. The genome size, heterozygosity and GC content obtained by k-mer analysis were almost 648.07 Mb, 0.72% and 36.11%, respectively. The genome size calculated by flow cytometry was 685.77 Mb, which was consistent with the results of genome survey. A total of 851,957 simple sequence repeats (SSR) were identified in the A. trifoliata genome. Twenty-eight phenotypic traits and thirty pairs of SSR primers were selected for the analysis of the genetic diversity of 43 accessions of cultivated A. trifoliata. The results showed that 216 bands were generated by 30 pairs of SSR primers, of which 189 (87.5%) were polymorphic. In addition, the phenotypes and SSR markers were used for cluster analysis of 43 cultivated accessions. The results of the two clustering methods were partially consistent. The genome survey of A. trifoliata demonstrated that the genome size of this plant was about 648.07 Mb. In the present study, the size and characteristics of the genome of A. trifoliata were reported for the first time, which greatly enriched the genomic resources of A. trifoliata for the further research and utilization.

A sweet protein monellin as a non-antibody scaffold for synthetic binding proteins.

Synthetic binding proteins that have the ability to bind with molecules can be generated using various protein domains as non-antibody scaffolds. These designer proteins have been used widely in research studies, as their properties overcome the disadvantages of using antibodies. Here, we describe the first application of a phage display to generate synthetic binding proteins using a sweet protein, monellin, as a non-antibody scaffold. Single-chain monellin (scMonellin), in which two polypeptide chains of natural monellin are connected by a short linker, has two loops on one side of the molecule. We constructed phage display libraries of scMonellin, in which the amino acid sequence of the two loops is diversified. To validate the performance of these libraries, we sorted them against the folding mutant of the green fluorescent protein variant (GFPuv) and yeast small ubiquitin-related modifier. We successfully obtained scMonellin variants exhibiting moderate but significant affinities for these target proteins. Crystal structures of one of the GFPuv-binding variants in complex with GFPuv revealed that the two diversified loops were involved in target recognition. scMonellin, therefore, represents a promising non-antibody scaffold in the design and generation of synthetic binding proteins. We termed the scMonellin-derived synthetic binding proteins 'SWEEPins'.

A dated phylogeny of Lardizabalaceae reveals an unusual long-distance dispersal across the Pacific Ocean and the rapid rise of East Asian subtropical evergreen broadleaved forests in the late Miocene.

Temperate South American-Asian disjunct distributions are the most unusual in organisms, and challenging to explain. Here, we address the origin of this unusual disjunction in Lardizabalaceae using explicit models and molecular data. The family (c.40 species distributed in ten genera) also provides an opportunity to explore the historical assembly of East Asian subtropical evergreen broadleaved forests, a typical and luxuriant vegetation in East Asia. DNA sequences of five plastid loci of 42 accessions representing 23 species of Lardizabalaceae (c. 57.5% of estimated species diversity), and 19 species from the six other families of Ranunculales, were used to perform phylogenetic analyses. By dating the branching events and reconstructing ancestral ranges, we infer that extant Lardizabalaceae dated to the Upper Cretaceous of East Asia and that the temperate South American lineage might have split from its East Asian sister group at c. 24.4 Ma. A trans-Pacific dispersal possibly by birds from East Asia to South America is plausible to explain the establishment of the temperate South American-East Asian disjunction in Lardizabalaceae. Diversification rate analyses indicate that net diversification rates of Lardizabalaceae experienced a significant increase around c. 7.5 Ma. Our findings suggest that the rapid rise of East Asian subtropical evergreen broadleaved forests occurred in the late Miocene, associated with the uplift of the Tibetan Plateau and the intensified East Asian monsoon, as well as the higher winter temperature and atmospheric CO2 levels.

Development of SSR markers via de novo transcriptome assembly in Akebia trifoliata (Thunb.) Koidz.

Owing to its high nutritive, economic, and medicinal values, Akebia trifoliata has received increased attention, making worthy of being used as a new fruit crop for further domestication and commercialization in China. However, molecular research of A. trifoliata has lagged as investigations of its genomic resources and molecular markers are rare. In this study, a cDNA library of A. trifoliata leaves was sequenced using the Illumina NovaSeq. 6000 sequencing system. In total, 101 417 transcripts, 63 757 unigenes, and 9494 simple sequence repeats were assembled and identified from the transcriptome datasets. The majority of the SSRs were di- and trinucleotide repeats. Length and number of SSR motifs ranged from 15 to 66, and 5 to 48 bp, respectively. Of which, the A/T mononucleotide motif and AG/TC and CT/GA dinucleotide motifs were the most abundant. Furthermore, 100 SSR primers were randomly selected to validate amplification and polymorphism, and 88 A. trifoliata accessions were definitively distinguished by 49 primers. With the Qinling mountains and Huaihe River line as the boundaries, the northern and southern accessions were clustered into different groups, but no clear geographical patterns (city or origin) were observed in the southern accessions. These newly identified molecular markers may provide a foundation for the genetic identification and diversity analysis and marker-assisted selection breeding in species of Akebia.

Complete Chloroplast Genome Sequence of Decaisnea insignis: Genome Organization, Genomic Resources and Comparative Analysis.

Decaisnea insignis is a wild resource plant and is used as an ornamental, medicinal, and fruit plant. High-throughput sequencing of chloroplast genomes has provided insight into the overall evolutionary dynamics of chloroplast genomes and has enhanced our understanding of the evolutionary relationships within plant families. In the present study, we sequenced the complete chloroplast genome of D. insignis and used the data to assess its genomic resources. The D. insignis chloroplast genome is 158,683 bp in length and includes a pair of inverted repeats of 26,167 bp that are separated by small and large single copy regions of 19,162 bp and 87,187 bp, respectively. We identified 83 simple sequence repeats and 18 pairs of large repeats. Most simple-sequence repeats were located in the noncoding sections of the large single-copy/small single-copy region and exhibited a high A/T content. The D. insignis chloroplast genome bias was skewed towards A/T on the basis of codon usage. A phylogenetic tree based on 82 protein-coding genes of 33 angiosperms showed that D. insignis was clustered with Akebia in Lardizabalaceae. Overall, the results of this study will contribute to better understanding the evolution, molecular biology and genetic improvement of D. insignis.