Biochemical Characterization and Functional Analysis of Glucose Regulated Protein 78 from the Silkworm Bombyx mori.

The glucose regulated protein (GRP78) is an important chaperone for various environmental and physiological stimulations. Despite the importance of GRP78 in cell survival and tumor progression, the information regarding GRP78 in silkworm Bombyx mori L. is poorly explored. We previously identified that GRP78 expression was significantly upregulated in the silkworm Nd mutation proteome database. Herein, we characterized the GRP78 protein from silkworm B. mori (hereafter, BmGRP78). The identified BmGRP78 protein encoded a 658 amino acid residues protein with a predicted molecular weight of approximately 73 kDa and comprised of two structural domains, a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). BmGRP78 was ubiquitously expressed in all examined tissues and developmental stages by quantitative RT-PCR and Western blotting analysis. The purified recombinant BmGRP78 (rBmGRP78) exhibited ATPase activity and could inhibit the aggregating thermolabile model substrates. Heat-induction or Pb/Hg-exposure strongly stimulated the upregulation expression at the translation levels of BmGRP78 in BmN cells, whereas no significant change resulting from BmNPV infection was found. Additionally, heat, Pb, Hg, and BmNPV exposure resulted in the translocation of BmGRP78 into the nucleus. These results lay a foundation for the future identification of the molecular mechanisms related to GRP78 in silkworms.

Development of 19 novel microsatellite markers of lily-of-the-valley (Convallaria, Asparagaceae) from transcriptome sequencing.

The lily-of-the-valley Convallaria (Asparagaceae) consists of three herbaceous perennial species. The plants are commonly found in northern hemisphere, and are best-known for their ornamental and pharmaceutical value. In order to assess the genetic structure, diversity and demographic history of Convallaria species, 19 novel microsatellite markers were developed based on transcriptome data of C. keiskei. Polymorphism and cross-amplification of the markers were tested in three populations of C. keiskei and one population each of C. majalis and C. montana. The transferability rate in two species was both 89.5%. The average number of alleles detected per locus was 7.7, 3.3 and 2.7 in C. keiskei, C. majalis and C. montana, respectively, and the polymorphism information content correspondingly varied from 0.067 to 0.730, from 0.071 to 0.637 and from 0.195 to 0.680 at the population level. The observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.833, respectively. Seven of the 19 loci showed significant deviation from Hardy-Weinberg equilibrium. The availability of these markers will provide a useful molecular tool for further population genetics, phylogeographic and breeding studies of Convallaria species.

Phylogenomic and Comparative Analyses of Complete Plastomes of Croomia and Stemona (Stemonaceae).

The monocot genus Croomia (Stemonaceae) comprises three herbaceous perennial species that exhibit EA (Eastern Asian)⁻ENA (Eastern North American) disjunct distribution. However, due to the lack of effective genomic resources, its evolutionary history is still weakly resolved. In the present study, we conducted comparative analysis of the complete chloroplast (cp) genomes of three Croomia species and two Stemona species. These five cp genomes proved highly similar in overall size (154,407⁻155,261 bp), structure, gene order and content. All five cp genomes contained the same 114 unique genes consisting of 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Gene content, gene order, AT content and IR/SC boundary structures were almost the same among the five Stemonaceae cp genomes, except that the Stemona cp genome was found to contain an inversion in cemA and petA. The lengths of five genomes varied due to contraction/expansion of the IR/SC borders. A/T mononucleotides were the richest Simple Sequence Repeats (SSRs). A total of 46, 48, 47, 61 and 60 repeats were identified in C. japonica, C. heterosepala, C. pauciflora, S. japonica and S. mairei, respectively. A comparison of pairwise sequence divergence values across all introns and intergenic spacers revealed that the ndhF⁻rpl32, psbM⁻trnD and trnS⁻trnG regions are the fastest-evolving regions. These regions are therefore likely to be the best choices for molecular evolutionary and systematic studies at low taxonomic levels in Stemonaceae. Phylogenetic analyses of the complete cp genomes and 78 protein-coding genes strongly supported the monophyly of Croomia. Two Asian species were identified as sisters that likely diverged in the Early Pleistocene (1.62 Mya, 95% HPD: 1.125⁻2.251 Mya), whereas the divergence of C. pauciflora dated back to the Late Miocene (4.77 Mya, 95% HPD: 3.626⁻6.162 Mya). The availability of these cp genomes will provide valuable genetic resources for further population genetics and phylogeographic studies on Croomia.

Genetic and geographical structure of boreal plants in their southern range: phylogeography of Hippuris vulgaris in China.

BACKGROUND: Our current understanding of the evolutionary history of boreal and arctic-alpine plants in their southern range in Asia remains relatively poor. Using three cpDNA non-coding regions and nine nuclear microsatellite (nSSR) loci, we examine the phylogeographic pattern in a broad geographic sampling of the boreal plant Hippuris vulgaris to infer its dispersal and diversification in China. In addition, the species distributions at the Last Glacial Maximum (LGM) and at present were predicted using ecological niche modeling (ENM). RESULTS: The cpDNA results revealed two distinct lineages, A and B. A is restricted to Northeast China; B is distributed in Northwest China, the Qinghai-Tibet Plateau (QTP) and North and Northeast (NNE) China; and A and B diverged ca. 1.36 Ma. The nSSR data revealed two genetic clusters corresponding to the two cpDNA lineages and nonreciprocal hybridization with lineage A as the maternal lineage in Northeast China. Cluster B further divided into three subclusters: I, mainly in NNE China and the northeastern border of the QTP; II, in Northwest China and the QTP; and III, on the QTP. ENM predicted a marked range shift on the QTP at the LGM, retreating from the platform to the northeast and southeast edges. CONCLUSIONS: Hippuris vulgaris probably diverged into lineages A and B in high latitudes and then immigrated into Northeast China and Northwest China, respectively. Lineage A persisted and diversified in Northeast China. Lineage B reached the QTP during the mid-Pleistocene, diversified in that region due to the influence of climatic oscillations, migrated into Northeast China and subsequently hybridized with lineage A. Our findings give empirical evidence that boreal plants display complex evolutionary history in their southern range in Asia and provide new insights into the evolution of boreal and arctic-alpine plants.

Comparative phylogeography of the wild-rice genus Zizania (Poaceae) in eastern Asia and North America.

PREMISE OF THE STUDY: Comparative phylogeography of intercontinental disjunct taxa allowed us not only to elucidate their diversification and evolution following geographic isolation, but also to understand the effect of climatic and geological histories on the evolutionary processes of closely related species. A phylogeographic analysis was conducted on the eastern Asian-North American disjunct genus Zizania to compare intracontinental phylogeographic patterns between different continents. METHODS: Surveys were conducted of 514 individuals using three chloroplast DNA fragments and three nuclear microsatellite loci. These individuals included 246 from 45 populations of Zizania latifolia in eastern Asia, and the following from North America: 154 individuals from 26 populations of Z. aquatica, 84 individuals from 14 populations of Z. palustris, and 30 individuals from one population of Z. texana. KEY RESULTS: The genetic diversity of North American Zizania was significantly higher than that of eastern Asian Zizania. High levels of genetic differentiation among populations and no signal of population expansion were detected in three widespread species. No phylogeographic structure was observed in Z. latifolia, and discordant patterns of cpDNA and microsatellite markers were observed in North American Zizania. CONCLUSIONS: Reduced variation in Zizania latifolia likely reflects its perennial life history, the North American origin of Zizania, and the relative homogeneity of aquatic environments. High levels of genetic differentiation suggest limited dispersal among populations in all Zizania species. The more complex patterns of diversification and evolution in North American Zizania may be driven by the greater impact of glaciation in North America relative to eastern Asia.

Effect of acute exposure of Hg on physiological parameters and transcriptome expression in silkworms (Bombyx mori).

Mercury (Hg) contamination poses a global threat to the environment, given its elevated ecotoxicity. Herein, we employed the lepidopteran model insect, silkworm (Bombyx mori), to systematically investigate the toxic effects of Hg-stress across its growth and development, histomorphology, antioxidant enzyme activities, and transcriptome responses. High doses of Hg exposure induced evident poisoning symptoms, markedly impeding the growth of silkworm larvae and escalating mortality in a dose-dependent manner. Under Hg exposure, the histomorphology of both the midgut and fat body exhibited impairments. Carboxylesterase (CarE) activity was increased in both midgut and fat body tissues responding to Hg treatment. Conversely, glutathione S-transferase (GST) levels increased in the fat body but decreased in the midgut. The transcriptomic analysis revealed that the response induced by Hg stress involved multiple metabolism processes. Significantly differently expressed genes (DEGs) exhibited strong associations with oxidative phosphorylation, nutrient metabolisms, insect hormone biosynthesis, lysosome, ribosome biogenesis in eukaryotes, and ribosome pathways in the midgut or the fat body. The findings implied that exposure to Hg might induce the oxidative stress response, attempting to compensate for impaired metabolism. Concurrently, disruptions in nutrient metabolism and insect hormone activity might hinder growth and development, leading to immune dysfunction in silkworms. These insights significantly advance our theoretical understanding of the potential mechanisms underlying Hg toxicity in invertebrate organisms.

Clear phylogeographical structures shed light on the origin and dispersal of the aquatic boreal plant Hippuris vulgaris.

Aquatic plants are an important ecological group in the arctic flora; however, their evolutionary histories remain largely unknown. In order to deepen our understanding of the evolution of these plants, we explored the phylogeographical structure of an aquatic boreal plant Hippuris vulgaris in a broad geographical sampling from Eurasia and North America using the chloroplast intergenic spacer psbA-trnH and seven nuclear microsatellite loci. Two closely-related species H. lanceolata and H. tetraphylla were also included because of their taxonomic controversy. Both chloroplast DNA sequences and nuclear microsatellite data revealed three genetic lineages with distinct distribution ranges. Incongruence between nuclear and chloroplast DNA lineages occurred in 14 samples from Russian Far East and Europe caused by inter-lineage hybridization. No private haplotypes or independent genetic clusters were evident in H. lanceolata or H. tetraphylla, suggesting that these two species should be considered conspecific ecotypes of H. vulgaris. Analysis using Approximate Bayesian Computation-Random Forest approach suggests that Hippuris vulgaris originated in China, followed by dispersal into Russia plus Northeast China, then successively westwards into Europe and North America, and finally into the Russian Far East from both North America and Russia plus Northeast China. This study is the first to elucidate the historical dispersal processes of a circumarctic aquatic plant across the entirety of its range.

The complete chloroplast genome of Sparganium angustifolium (Typhaceae).

The complete chloroplast (cp) genome of Sparganium angustifolium was sequenced and annotated in the present study. The circular genome is 161,720 bp in length and exhibits a typical quadripartite structure with a large single-copy (LSC, 88,981 bp) and small single-copy (SSC, 18,731 bp) regions, separated by a pair of inverted repeats (IRs, 27,004 bp). The cp genome contains 114 unique genes, including 80 protein-coding, 30 tRNA, and four rRNA genes. The phylogenetic analysis of Typhaceae strongly supported the monophyly of Sparganium and resolved two clades that represented newly revised two subgenera. S. angustifolia has the closest relationship with S. emersum in the present sampling extent.

Evolutionary Comparison of the Complete Chloroplast Genomes in Convallaria Species and Phylogenetic Study of Asparagaceae.

The genus Convallaria (Asparagaceae) comprises three herbaceous perennial species that are widely distributed in the understory of temperate deciduous forests in the Northern Hemisphere. Although Convallaria species have high medicinal and horticultural values, studies related to the phylogenetic analysis of this genus are few. In the present study, we assembled and reported five complete chloroplast (cp) sequences of three Convallaria species (two of C. keiskei Miq., two of C. majalis L., and one of C. montana Raf.) using Illumina paired-end sequencing data. The cp genomes were highly similar in overall size (161,365-162,972 bp), and all consisted of a pair of inverted repeats (IR) regions (29,140-29,486 bp) separated by a large single-copy (LSC) (85,183-85,521 bp) and a small single-copy (SSC) region (17,877-18,502 bp). Each cp genome contained the same 113 unique genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Gene content, gene order, AT content and IR/SC boundary structure were nearly identical among all of the Convallaria cp genomes. However, their lengths varied due to contraction/expansion at the IR/LSC borders. Simple sequence repeat (SSR) analyses indicated that the richest SSRs are A/T mononucleotides. Three highly variable regions (petA-psbJ, psbI-trnS and ccsA-ndhD) were identified as valuable molecular markers. Phylogenetic analysis of the family Asparagaceae using 48 cp genome sequences supported the monophyly of Convallaria, which formed a sister clade to the genus Rohdea. Our study provides a robust phylogeny of the Asparagaceae family. The complete cp genome sequences will contribute to further studies in the molecular identification, genetic diversity, and phylogeny of Convallaria.

Characterization of the complete chloroplast genome of Sparganium glomeratum (Typhaceae) from Jilin Province, China and phylogenetic analysis.

The complete chloroplast genome of Sparganium glomeratum was sequenced and assembled in this study. The circular genome is 160,391 bp in length and exhibits a typical quadripartite structure with a large single-copy (LSC, 87,660 bp) and small single-copy (SSC, 18,721 bp) regions, separated by a pair of inverted repeats (IRs, 27,005 bp). The cp genome contains 113 unique genes, including 79 protein-coding, 30 tRNA, and four rRNA genes. The phylogenetic analysis within the Poales showed that Sparganium is monophyletic and most closely related to Typha. Within Sparganium, S. glomeratum is sister to the clade of S. stoloniferum and S. euricarpum. The work reported here will provide useful information for the evolutionary studies on the genus of Sparganium.

The complete chloroplast genome sequences of Hippuris vulgaris (Plantaginaceae).

Two complete chloroplast genomes of Hippuris vulgaris (H. vulgaris_A and H. vulgaris_B), representing two distinct clades in China, were sequenced and assembled in this study. The circular genomes were 152,763 and 152,713 bp in length and exhibit a typical quadripartite structure of the large single-copy (LSC, 82,983/82,949 bp) and small single-copy (SSC, 18,294/18,278 bp) regions, separated by a pair of inverted repeats (IRs, both 25,743 bp). Both two cp genomes identically contain 133 genes, including 88 protein-coding genes, 37 tRNA, and eight rRNA genes. The phylogenetic analysis within Plantaginaceae demonstrated Hippuris an independent clade included in the expanded Plantaginaceae.

Generation and classification of transcriptomes in two Croomia species and molecular evolution of CYC/TB1 genes in Stemonaceae.

The genus Croomia (Stemonaceae) is an excellent model for studying the evolution of the Eastern Asia (EA)-Eastern North America (ENA) floristic disjunction and the genetic mechanisms of floral zygomorphy formation. In addition to the presence of both actinomorphic and zygomorphic flowers within the genus, species are disjunctively distributed between EA and ENA. However, due to the limited availability of genomic resources, few studies of Croomia have examined these questions. In this study, we sequenced the floral and leaf transcriptomes of the zygomorphic flowered C roomia heterosepala and the actinomorphic flowered Croomia japonica, and used comparative genomic approaches to investigate the transcriptome evolution of the two closely related species. The sequencing and de novo assembly of transcriptomes from flowers of C. heterosepala (ChFlower), flowers of C. japonica (CjFlower), and leaves of C. japonica (CjLeaf) yielded 57,193, 62,131 and 64,448 unigenes, respectively. In addition, estimation of Ka/Ks ratios for 11,566 potential orthologous groups between ChFlower and CjFlower revealed that only six pairs had Ka/Ks ratios significantly greater than 1 and are likely under positive selection. A total of 429 single copy nuclear genes (SCNGs) and 21,460 expression sequence tags-simple sequence repeats (EST-SSRs) were identified in this study. Specifically, we identified seven CYC/TB1-like genes from Stemonaceae. Phylogenetic and molecular evolution analyses indicated that these CYC/TB1-like genes formed a monophyletic clade (SteTBL1) and were subject to strong purifying selection. The shifts of floral symmetry in Stemonaceae do not appear to be correlated with TBL copy number.