Complete chloroplast genomes of Rubus species (Rosaceae) and comparative analysis within the genus.

BACKGROUND: Rubus is the largest genus of the family Rosaceae and is valued as medicinal, edible, and ornamental plants. Here, we sequenced and assembled eight chloroplast (cp) genomes of Rubus from the Dabie Mountains in Central China. Fifty-one Rubus species were comparatively analyzed for the cp genomes including the eight newly discovered genomes and forty-three previously reported in GenBank database (NCBI). RESULTS: The eight newly obtained cp genomes had the same quadripartite structure as the other cp genomes in Rubus. The length of the eight plastomes ranged from 155,546 bp to 156,321 bp with similar GC content (37.0 to 37.3%). The results indicated 133-134 genes were annotated for the Rubus plastomes, which contained 88 or 89 protein coding genes (PCGs), 37 transfer RNA genes (tRNAs), and eight ribosomal RNA genes (rRNAs). Among them, 16 (or 18) of the genes were duplicated in the IR region. Structural comparative analysis results showed that the gene content and order were relatively preserved. Nucleotide variability analysis identified nine hotspot regions for genomic divergence and multiple simple sequences repeats (SSRs), which may be used as markers for genetic diversity and phylogenetic analysis. Phylogenetic relationships were highly supported within the family Rosaceae, as evidenced by sub-clade taxa cp genome sequences. CONCLUSION: Thus, the whole plastome may be used as a super-marker in phylogenetic studies of this genus.

Maintenance of species boundaries in three sympatric Ligularia (Senecioneae, Asteraceae) species.

The key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages. Although species boundaries are frequently investigated between two species across many taxa, reproductive isolating barriers among multiple species (>2) that would represent the most common phenomenon in nature, remain to be clarified. Here, we use double digest restriction-site associated DNA (ddRAD) sequencing to examine patterns of hybridization at a sympatric site where three Ligularia species grow together and verify whether those patterns contribute to the maintenance of boundaries among species. The results based on the RAD SNP datasets indicated hybridization Ligularia cyathiceps × L. duciformis and L. duciformis × L. yunnanensis were both restricted to F1 s plus a few first-generation backcrosses and no gene introgression were identified, giving rise to strong reproductive isolation among hybridizing species. Moreover, hybrid swarm simulation, using HYBRIDLAB, indicated the RAD SNP datasets had sufficient discriminatory power for accurate hybrid detection. We conclude that parental species show strong reproductive isolation and they still maintain species boundaries, which may be the key mechanism to maintain species diversity of Ligularia in the eastern Qinghai-Tibetan Plateau and adjacent areas. Moreover, this study highlights the effectiveness of RAD sequencing in hybridization studies.

Chemical Constituents in Hybrids of Ligularia tongolensis and L. cymbulifera: Chemical Introgression in L. tongolensis.

Two samples with morphologies intermediate between Ligularia tongolensis and L. cymbulifera were collected in Desha, Sichuan Province, and one, in Pachahai, Yunnan Province, P. R. China. The DNA sequencing confirmed that the samples were hybrids of the two species. Tetradymol (1), the major compound of L. cymbulifera not found in L. tongolensis, was isolated from the hybrid samples collected at both locations, while furanoeremophilan-15-oic acid derivative 4, a compound characteristic to L. tongolensis, was found in the Pachahai hybrid but not in the Desha hybrids. Thus, the chemical consequence of hybridization can be variable. In addition, analysis of L. tongolensis samples at Pachahai indicated that introgression has been a mechanism of generating chemical diversity in the plant. Eleven compounds including three new ones were isolated.

De novo genome assembly of endophytic fungus Nemania diffusa strain YAFEF818, isolated from Artemisia argyi.

Here, we report a draft genome sequence of endophytic fungus Nemania diffusa YAFEF818, isolated from Artemisia argyi. Oxford Nanopore Technologies PromethION and Illumina NovaSeq sequence reads were assembled using NECAT and polished using pilon to yield a 55.63 Mb genome.

Characterization of the complete chloroplast genome sequence of Limnophila sessiliflora Blume 1826 (Plantaginaceae).

Limnophila sessiliflora Blume 1826 is a perennial amphibious herb with ornamental and water purification value that is widespread in temperate and tropical Asia. In the present study, we sequenced, assembled, and annotated the complete chloroplast (cp) genome of L. sessiliflora. It is 152,395 bp in length, with a typical quadripartite structure, comprising a pair of inverted repeat regions (IRs; 25,545 bp), a large single-copy region (LSC; 83,163 bp), and a small single-copy (SSC; 18,142 bp) region. The whole cp genome contained 135 genes, including 89 protein-coding genes (PCGs), 38 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The maximum-likelihood (ML) phylogenetic analysis indicated that L. sessiliflora was closely related to the genera Bacopa and Scoparia in the tribe Gratioleae of Plantaginaceae. This cp genome provides a valuable genetic resource for phylogenetic study.

Genomic Analysis of the Xanthoria elegans and Polyketide Synthase Gene Mining Based on the Whole Genome.

Xanthoria elegans is a lichen symbiosis, that inhabits extreme environments and can absorb UV-B. We reported the de novo sequencing and assembly of X. elegans genome. The whole genome was approximately 44.63 Mb, with a GC content of 40.69%. Genome assembly generated 207 scaffolds with an N50 length of 563,100 bp, N90 length of 122,672 bp. The genome comprised 9,581 genes, some encoded enzymes involved in the secondary metabolism such as terpene, polyketides. To further understand the UV-B absorbing and adaptability to extreme environments mechanisms of X. elegans, we searched the secondary metabolites genes and gene-cluster from the genome using genome-mining and bioinformatics analysis. The results revealed that 7 NR-PKSs, 12 HR-PKSs and 2 hybrid PKS-PKSs from X. elegans were isolated, they belong to Type I PKS (T1PKS) according to the domain architecture; phylogenetic analysis and BGCs comparison linked the putative products to two NR-PKSs and three HR-PKSs, the putative products of two NR-PKSs were emodin xanthrone (most likely parietin) and mycophelonic acid, the putative products of three HR-PKSs were soppilines, (+)-asperlin and macrolactone brefeldin A, respectively. 5 PKSs from X. elegans build a correlation between the SMs carbon skeleton and PKS genes based on the domain architecture, phylogenetic and BGC comparison. Although the function of 16 PKSs remains unclear, the findings emphasize that the genes from X. elegans represent an unexploited source of novel polyketide and utilization of lichen gene resources.

Small RNA sequencing provides insights into molecular mechanism of flower development in Rhododendron pulchrum Sweet.

Rhododendron pulchrum sweet, a member of the Ericaceae family possessing valuable horticultural properties, is widely distributed in the temperate regions. Though serving as bioindicator of metal pollution, the molecular mechanism regulating flowering in R. pulchrum is very limited. Illumina sequencing was performed to identify critical miRNAs in the synthesis of flavonoids at different developmental stages. Totally, 722 miRNAs belonging to 104 families were screened, and 84 novel mature miRNA sequences were predicted. The miR166, miR156, and miR167-1 families were dominant. In particular, 126 miRNAs were significantly differentially expressed among four different flowering stages. Totally, 593 genes were differentially regulated by miRNAs during the flower development process, which were mostly involved in "metabolic pathways", "plant hormone signal transduction", and "mitosis and regulation of biosynthetic processes". In pigment biosynthesis and signal transduction processes, gra-miR750 significantly regulated the expression of flavonoid 3',5'-hydroxylase; aof-miR171a, aof-miR171b, aof-miR171c, cas-miR171a-3p, and cas-miR171c-3p could regulate the expression of DELLA protein; aof-miR390, aof-miR396b, ath-miR3932b-5p, cas-miR171a-3p, aof-miR171a, and aof-miR171b regulated BAK1 expression. This research showed great potentials for genetic improvement of flower color traits for R. pulchrum and other Rhododendron species.

Complete chloroplast genome sequence of Rhododendronmariesii and comparative genomics of related species in the family Ericaeae.

Rhododendronmariesii Hemsley et Wilson, 1907, a typical member of the family Ericaeae, possesses valuable medicinal and horticultural properties. In this research, the complete chloroplast (cp) genome of R.mariesii was sequenced and assembled, which proved to be a typical quadripartite structure with the length of 203,480 bp. In particular, the lengths of the large single copy region (LSC), small single copy region (SSC), and inverted repeat regions (IR) were 113,715 bp, 7,953 bp, and 40,918 bp, respectively. Among the 151 unique genes, 98 were protein-coding genes, 8 were tRNA genes, and 45 were rRNA genes. The structural characteristics of the R.mariesiicp genome was similar to other angiosperms. Leucine was the most representative amino acid, while cysteine was the lowest representative. Totally, 30 codons showed obvious codon usage bias, and most were A/U-ending codons. Six highly variable regions were observed, such as trnK-pafI and atpE-rpoB, which could serve as potential markers for future barcoding and phylogenetic research of R.mariesii species. Coding regions were more conserved than non-coding regions. Expansion and contraction in the IR region might be the main length variation in R.mariesii and related Ericaeae species. Maximum-likelihood (ML) phylogenetic analysis revealed that R.mariesii was relatively closed to the R.simsii Planchon, 1853 and R.pulchrum Sweet,1831. This research will supply rich genetic resource for R.mariesii and related species of the Ericaeae.

The complete mitochondrial genome of a polyphagous insect: Colasposoma dauricum (Coleoptera: Chrysomelidae: Eumolpinae).

Colasposoma dauricum Mannerheim, 1849, is an important insect pest distributed in most areas of China. The complete mitochondrial genome of C. dauricum was sequenced and analyzed. The phylogenetic relationships between C. dauricum and other 10 species in the superfamily Chrysomeloidea were reconstructed using maximum likelihood (ML) methods based on the concatenated nucleotide sequences, the phylogenetic analysis showed that C. dauricum is closely related to Basilepta fulvipes in the same subfamily.

The complete chloroplast genome sequence of Callitriche palustris (Plantaginaceae).

Callitriche palustris L. is an annual aquatic or marsh plant, wide spread in temperate regions throughout the world. In present study, we sequenced, assembled and annotated the complete chloroplast (cp) genome of C. palustris. The length of C. palustris complete cp genome was 150,138 bp, with a typical quadripartite structure comprising a pair of inverted repeat regions (IRs; 25,667 bp), a large single copy region (LSC; 81,432 bp) and a small single copy region (SSC; 17,372 bp). The whole cp genome contained 134 genes, including 89 protein-coding genes (PCGs), 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The maximum likelihood (ML) phylogenetic analysis indicated that C. palustris was a member of Plantaginaceae, but the relationships between subfamilies and tribes need more samplings. This cp genome would provide a valuable genetic resource for C. palustris' phylogenetic study.

The complete chloroplast genome sequence of China Lindera praecox (Lauraceae) and intra-species diversity.

Lindera praecox is a signature composition in the broadleaved deciduous forest of East China and Japan. Presently, the complete chloroplast (cp) genome of this species was sequenced, assembled, and annotated. It is 152,818 bp in length and encodes 85 protein-coding genes, 36 transfer RNA (tRNA) genes and eight ribosomal RNA (rRNA) genes. The phylogenetic analysis indicated intraspecific varieties within L. praecox species collected in China and Japan. This chloroplast genome sequencing offers genetic background for resources conservation and phylogenetic studies.

The complete chloroplast genome sequence of Sinojackia huangmeiensis (Styracaceae).

Sinojackia huangmeiensis J. W. Ge & X. H. Yao is a member of the genus Sinojackia endemic to central and east China. Here we assembled and annotated the complete chloroplast (cp) genome. It is 158,758 bp in length and encodes 84 protein-coding genes, 37 transfer RNA (tRNA) genes and eight ribosomal RNA (rRNA) genes. The Maximum likelihood phylogenetic analysis confirm that S. huangmeiensis is a closely related but another different species to S. sarcocarpa.

The complete chloroplast genome sequence of Anogeissus acuminata (combretaceae).

Anogeissus acuminata (Roxburgh ex Candolle) Guillemin et al. is an Endangered and dominant species of deciduous forests distributed in the Mekong valley of southwest China and adjacent Indo-China Peninsula. Here we assembled and annotated the complete chloroplast (cp) genome. It is 159,993 bp in length and encodes 84 protein-coding genes, 37 transfer RNA (tRNA) genes and eight ribosomal RNA (rRNA) genes. This chloroplast genome sequencing offers genetic background for conservation and phylogenetic studies.

The complete chloroplast genome sequence of Brainea insignis (Blechnaceae).

Brainea insignis (Hooker) J. Smith, a member of Blechnaceae, is a rare and endangered species in tropical Asia. Here we assembled and annotated the complete chloroplast (cp) genome. It is 149,730 bp in length and encodes 88 protein-coding genes, 36 transfer RNA (tRNA) genes and eight ribosomal RNA (rRNA) genes. This chloroplast genome sequencing offers a useful resource for future conservation genetics and phylogenetic studies.

Complete chloroplast genome sequence and phylogenetic analysis of Magnolia pilocarpa, a highly ornamental species endemic in central China.

Magnolia pilocarpa Z. Z. Zhao et Z. W. Xie is a species with high horticultural and medicinal value, which found only in Dabie Mountain of central China. In this study, we sequenced, assembled and annotated the complete chloroplast (cp) genome of M. pilocarpa. The length of M. pilocarpa complete cp genome was 160,104 bp, with a typical quadripartite structure comprising a pair of inverted repeat regions (IRs; 26,591 bp), a large single copy region (LSC; 88,110 bp) and a small single copy region (SSC; 18,812 bp). The whole cp genome contained 129 unique genes, including 79 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes and four ribosomal RNA (rRNA) genes. The maximum-likelihood (ML) phylogenetic analysis indicated that M. pilocarpa was relatively closed to the M. denudata. This chloroplast genome would provide a valuable genetic resource for M. pilocarpa.

Complete mitochondrial genome of Niphades castanea (Coleoptera: Curculionidae).

Niphades castanea Chao is an important insect pest on many plants which belong to genus Castanea. The complete mitochondrial genome of N. castanea was sequenced and analyzed. The phylogenetic relationships between N. castanea and other 41 species in the family Curculionidae were reconstructed using maximum likelihood (ML) methods based on the concatenated nucleotide sequences, the phylogenetic analysis showed that N. castanea is closely related to Hylobitelus xiaoi, which is in accordance with the traditional morphological classification.

The complete chloroplast genome sequence of Melica scabrosa (Poaceae).

Melica scabrosa Trin. is an important forage grass of Poaceae, wildly distributed in the Northeast Asia to Qinghai-Xizang Plateau. The complete chloroplast genome sequence of M. scabrosa was obtained by de novo assembly using whole genome sequence data. The chloroplast genome is 134,889 bp in length, containing 80,560 bp in a large single copy (LSC), 12,706 bp in a small single copy (SSC) and 20,810 bp in a pair of inverted repeats (IRs). A total of 129 genes including 83 protein-coding genes and 38 structural RNA genes were identified. Phylogenetic analysis represented close relationship among Melica species. This chloroplast genome sequencing offers a useful resource for future genetics and phylogenetic studies.

Molecular evidence for asymmetric hybridization in three closely related sympatric species.

Natural hybridization is common in plants and results in different evolutionary consequences to hybridizing species. Pre- and post-zygotic reproductive isolating barriers can impede hybridization between closely related species to maintain their species integrity. In Northwest Yunnan, three Ligularia species (Ligularia cyathiceps, L. duciformis and L. yunnanensis) and four types of morphologically intermediate individuals were discovered growing together in an area subject to human disturbance. In this study, we used three low-copy nuclear loci to test the natural hybridization hypothesis and the hybridization direction was ascertained by three chloroplast DNA fragments. The results indicated there were two hybridization groups at the study site, L. cyathiceps × L. duciformis and L. duciformis × L. yunnanensis, and two types of morphologically intermediate individuals were produced by L. cyathiceps and L. duciformis, and another two types were produced by L. duciformis and L. yunnanensis, while no hybrids between L. cyathiceps and L. yunnanensis were observed. Both hybridizing groups showed bidirectional but asymmetric hybridization and the factors influencing the symmetry are discussed. Most hybrids produced by the two hybridization groups seemed to be F1 generation. Hybrids with different morphologies within the same hybridization group showed similar genetic components. The results suggest that although human disturbance may promote natural hybridization among the three Ligularia species bringing them together, hybrids are limited to F1s and therefore species boundaries might be maintained.

Degeneration of photosynthetic capacity in mixotrophic plants, Chimaphila japonica and Pyrola decorata (Ericaceae).

The evolution of photosynthesis is an important feature of mixotrophic plants. Previous inferences proposed that mixotrophic taxa tend to retain most genes relating to photosynthetic functions but vary in plastid gene content. However, no sequence data are available to test this hypothesis in Ericaceae. To investigate changes in plastid genomes that may result from a transition from autotrophy to mixotrophy, the plastomes of two mixotrophic plants, Pyrola decorata and Chimaphila japonica, were sequenced at Illumina's Genome Analyzer and compared to the published plastome of the autotrophic plant Rhododendron simsii, which also belongs to Ericaceae. The greatest discrepancy between mixotrophic and autotrophic plants was that ndh genes for both P. decorata and C. japonica plastomes have nearly all become pseudogenes. P. decorata and C. japonica also retained all genes directly involved in photosynthesis under strong selection. The calculated rate of nonsynonymous nucleotide substitutions and synonymous substitutions of protein-coding genes (dN/dS) showed that substitution rates in shade plants were apparently higher than those in sunlight plants. The two mixotrophic plastomes were generally very similar to that of non-parasitic plants, although ndh genes were largely pseudogenized. Photosynthesis genes under strong selection were retained in the two mixotrophs, however, with greatly increased substitution rates. Further research is needed to gain a clearer understanding of the evolution of autotrophy and mixotrophy in Ericaceae.

Bidirectional natural hybridization between sympatric Ligularia vellerea and L. subspicata.

Natural hybridization has been regarded as a crucial pathway of speciation and provides the raw materials for the evolution of biodiversity. The interspecific natural hybridization of the genus Ligularia Cass. is universal and has been considered to be an important factor driving the high diversity of Ligularia species in the Hengduan Mountains, China. Although the natural hybridization between L. vellerea and L. subspicata was reported previously, the direction of hybridization was uncertain due to the limitation of sampling. Thus, in this study, we sampled more individuals and increased two fragments of chloroplast DNA on the basis of the previous study to further verify the natural hybridization between L. vellerea and L. subspicata and confirm the direction of hybridization. Based on DNA sequences (atpB-rbcL, trnL-rpl32, trnQ-5'rps16, and nuclear ribosomal internal transcribed spacer region) data, we concluded that putative hybrids were primary products of hybridization between L. vellerea and L. subspicata and the hybridization was bidirectional. Moreover, sympatric L. tongolensis was not apparently involved in the hybridization. Surprisingly, some pure L. subspicata individuals showed the disaccordance between morphology and DNA data, which might indicate that introgression occurs between L. vellerea and L. subspicata.