Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome.

Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.

Generation and Application of Mouse-Rat Allodiploid Embryonic Stem Cells.

Mammalian interspecific hybrids provide unique advantages for mechanistic studies of speciation, gene expression regulation, and X chromosome inactivation (XCI) but are constrained by their limited natural resources. Previous artificially generated mammalian interspecific hybrid cells are usually tetraploids with unstable genomes and limited developmental abilities. Here, we report the generation of mouse-rat allodiploid embryonic stem cells (AdESCs) by fusing haploid ESCs of the two species. The AdESCs have a stable allodiploid genome and are capable of differentiating into all three germ layers and early-stage germ cells. Both the mouse and rat alleles have comparable contributions to the expression of most genes. We have proven AdESCs as a powerful tool to study the mechanisms regulating X chromosome inactivation and to identify X inactivation-escaping genes, as well as to efficiently identify genes regulating phenotypic differences between species. A similar method could be used to create hybrid AdESCs of other distantly related species.

A lncRNA from the FTO locus acts as a suppressor of the m6A writer complex and p53 tumor suppression signaling.

N6-methyladenosine (m6A) of mRNAs modulated by the METTL3-METTL14-WTAP-RBM15 methyltransferase complex and m6A demethylases such as FTO play important roles in regulating mRNA stability, splicing, and translation. Here, we demonstrate that FTO-IT1 long noncoding RNA (lncRNA) was upregulated and positively correlated with poor survival of patients with wild-type p53-expressing prostate cancer (PCa). m6A RIP-seq analysis revealed that FTO-IT1 knockout increased mRNA m6A methylation of a subset of p53 transcriptional target genes (e.g., FAS, TP53INP1, and SESN2) and induced PCa cell cycle arrest and apoptosis. We further showed that FTO-IT1 directly binds RBM15 and inhibits RBM15 binding, m6A methylation, and stability of p53 target mRNAs. Therapeutic depletion of FTO-IT1 restored mRNA m6A level and expression of p53 target genes and inhibited PCa growth in mice. Our study identifies FTO-IT1 lncRNA as a bona fide suppressor of the m6A methyltransferase complex and p53 tumor suppression signaling and nominates FTO-IT1 as a potential therapeutic target of cancer.

N6-Deoxyadenosine Methylation in Mammalian Mitochondrial DNA.

N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.

Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm.

FTO, the first RNA demethylase discovered, mediates the demethylation of internal N6-methyladenosine (m6A) and N6, 2-O-dimethyladenosine (m6Am) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m6A and cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am in snRNAs, and N1-methyladenosine (m1A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m6A than the ones with cap m6Am in the tested cells. We also show that FTO can directly repress translation by catalyzing m1A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m6A and m6Am in mRNA and snRNA as well as m1A in tRNA.

5-Hydroxymethylcytosine signals in serum are a predictor of chemoresistance in high-grade serous ovarian cancer.

OBJECTIVE: The genome-wide profiling of 5-hydroxymethylcytosines (5hmC) on circulating cell-free DNA (cfDNA) has revealed promising biomarkers for various diseases. The purpose of this study was to investigate 5hmC signals in serum cfDNA and identify novel predictive biomarkers for the development of chemoresistance in high-grade serous ovarian cancer (HGSOC). We hypothesized that 5hmC profiles in cfDNA reflect the development of chemoresistance and elucidate pathways that may drive chemoresistance in HGSOC. Moreover, we sought to identify predictors that would better stratify outcomes for women with intermediate-sensitive HGSOC. METHODS: Women diagnosed with HGSOC and known platinum sensitivity status were selected for this study. Nano-hmC-Seal was performed on cfDNA isolated from archived serum samples, and differential 5hmC features were identified using DESeq2 to establish a model predictive of chemoresistance. RESULTS: A multivariate model consisting of three features (preoperative CA-125, largest residual implant after surgery, 5hmC level of OSGEPL), stratified samples from intermediate sensitive, chemo-naive women diagnosed with HGSOC into chemotherapy-resistant- and sensitive-like strata with a significant difference in overall survival (OS). Independent analysis of The Cancer Genome Atlas data further confirmed that high OSGEPL1 expression is a favorable prognostic factor for HGSOC. CONCLUSIONS: We have developed a novel multivariate model based on clinico-pathologic data and a cfDNA-derived 5hmC modified gene, OSGEPL1, that predicted response to platinum-based chemotherapy in intermediate-sensitive HGSOC. Our multivariate model applies to chemo-naïve samples regardless if the patint was treated with adjuvant or neoadjuvant chemotherapy. These results merit further investigation of the predictive capability of our model in larger cohorts.

METTL14 facilitates global genome repair and suppresses skin tumorigenesis.

Global genome repair (GGR), a subpathway of nucleotide excision repair, corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here, we show that METTL14 (methyltransferase-like 14), a critical component of the N6-methyladenosine (m6A) RNA methyltransferase complex, promotes GGR through regulating m6A mRNA methylation-mediated DDB2 translation and suppresses ultraviolet B (UVB) radiation-induced skin tumorigenesis. UVB irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy. METTL14 knockdown decreases GGR and DDB2 abundance. Conversely, overexpression of wild-type METTL14 but not its enzymatically inactive mutant increases GGR and DDB2 abundance. METTL14 knockdown decreases m6A methylation and translation of the DDB2 transcripts. Adding DDB2 reverses the GGR repair defect in METTL14 knockdown cells, indicating that METTL14 facilitates GGR through regulating DDB2 m6A methylation and translation. Similarly, knockdown of YTHDF1, an m6A reader promoting translation of m6A-modified transcripts, decreases DDB2 protein levels. Both METTL14 and YTHDF1 bind to the DDB2 transcript. In mice, skin-specific heterozygous METTL14 deletion increases UVB-induced skin tumorigenesis. Furthermore, METTL14 as well as DDB2 is down-regulated in human and mouse skin tumors and by chronic UVB irradiation in mouse skin, and METTL14 level is associated with the DDB2 level, suggesting a tumor-suppressive role of METTL14 in UVB-associated skin tumorigenesis in association with DDB2 regulation. Taken together, these findings demonstrate that METTL14 is a target for selective autophagy and acts as a critical epitranscriptomic mechanism to regulate GGR and suppress UVB-induced skin tumorigenesis.

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation.

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2'O-methylation, pseudouridylation, N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m6A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m6A modification, the methyltransferase responsible for the 18S rRNA m6A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m6A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m6A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m6A in noncoding RNAs.

A critical role of nuclear m6A reader YTHDC1 in leukemogenesis by regulating MCM complex-mediated DNA replication.

YTHDC1 has distinct functions as a nuclear N6-methyladenosine (m6A) reader in regulating RNA metabolism. Here we show that YTHDC1 is overexpressed in acute myeloid leukemia (AML) and that it is required for the proliferation and survival of human AML cells. Genetic deletion of Ythdc1 markedly blocks AML development and maintenance as well as self-renewal of leukemia stem cells (LSCs) in vivo in mice. We found that Ythdc1 is also required for normal hematopoiesis and hematopoietic stem and progenitor cell (HSPC) maintenance in vivo. Notably, Ythdc1 haploinsufficiency reduces self-renewal of LSCs but not HSPCs in vivo. YTHDC1 knockdown has a strong inhibitory effect on proliferation of primary AML cells. Mechanistically, YTHDC1 regulates leukemogenesis through MCM4, which is a critical regulator of DNA replication. Our study provides compelling evidence that shows an oncogenic role and a distinct mechanism of YTHDC1 in AML.

Azospirillum Aestuarii sp. nov., a Novel Nitrogen-Fixting and Aerobic Denitrifying Bacteria Isolated from an Estuary of a Freshwater River.

A novel Gram-staining negative, aerobic, rod-shaped bacterium, designated strain YIM DDC1T, was isolated from an estuary sediment sample of Dongda River flowing into Dianchi lake in Yunnan, southwest China. The strain displayed growth at 10-40 °C (optimum of 28 °C), pH 5.0-9.0 (optimum of 7.0-8.0) and in presence of 0-3% (w/v) NaCl (optimum of 0-1%). Strain YIM DDC1T comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and two unidentified aminolipids as the predominant polar lipids; the ubiquinone 10 as the major respiratory quinone; and summed feature 8 (C18:1ω6c and/or C18:1ω7c), summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C18:1 2-OH as the major cellular fatty acids. Analysis of 16S rRNA showed that YIM DDC1T represents a member of the genus Azospirillum, and was closely related to A. brasilense ATCC 29145 T (98.9%), A. baldaniorum Sp245T (98.2%), A. argentinense Az39T (98.2%) and A. formosense CC-Nfb-7 T (98.2%). The draft genome size was 7.15 Mbp with a 68.4% G + C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain YIM DDC1T and the aforementioned closely related strains exhibited similarity in the range of 93.8-93.5% and 53.7-52.7%, respectively. nif gene cluster (nifHDK) and denitrification genes ((napA, nirS, nirK, norBC and nosZ) detected in the genome indicated its potential nitrogen fixation and full-fledged denitrifying function. Based on combined genotypic and phenotypic data, strain YIM DDC1T represents a novel species of the genus Azospirillum, for which the name Azospirillum aestuarii sp. nov. is proposed. The type strain is YIM DDC1T (= KCTC 42887 T = CGMCC 1.17325 T).

Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response.

Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.

Evolution of a reverse transcriptase to map N1-methyladenosine in human messenger RNA.

Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m1A sites and revealed hundreds of m1A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m1A biology.

A metabolic labeling method detects m6A transcriptome-wide at single base resolution.

Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resolution remains an issue, impeding our understanding of m6A roles at the nucleotide level. Here, we report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resolution, called 'm6A-label-seq'. Human and mouse cells could be fed with a methionine analog, Se-allyl-L-selenohomocysteine, which substitutes the methyl group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. We pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. We identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications.

First Report of Anthracnose Crown Rot on Strawberry (Fragaria × ananassa Duch.) Caused by Colletotrichum pandanicola in Yunnan Provence, China.

Strawberry (Fragaria × ananassa Duch.), a widely grown octoploid species, is one of the most important economic fruit crops and has been widely cultivated in the world, including China. In December 2021, a serious crown rot disease (approximately 50% incidence) was observed in strawberry (cultivar Miaoxiang) plantations in Qujing City, Yunnan Province, China. Symptoms observed on aboveground part withered rapidly, reddish-brown marbled necrosis on crown. The roots were healthy and strong, but the plants finally died. To isolate the causal agent of this disease, crown tissues from five strawberry plants showing typical symptoms were cut into pieces of 5×5 mm, and the pieces were surface-sterilized with 75% ethanol for 45 s followed by 2.5% NaClO for 3 min and rinsed thrice with sterile water, and then placed onto potato dextrose agar (PDA) for 7 days at 25 ºC. After 3 to 4 days, extended single hyphal tips from the tissues were transferred to PDA and incubated for 7 days at 25 ºC. The colonies were initially white, later became somewhat zonate, velvety, cyan gray on the upper side and cyan ink pigment ring on the reverse side of plates, with concentric rings of salmon sporodochia. Many yellowish or orange creamy conidial droplets formed on PDA after 14 days at 25 ºC. Fifty-nine isolates were obtained, and three isolates QLYRR1, QLMCR9, and QLMCR39 were selected for further experiments. Conidia were hyaline, cylindrical with rounded ends, 12.17-19.35×3.71-6.30 µm (average±SD, 15.24±1.37×5.09±0.45 µm, n=150), L/W ratio = 2.99. The three isolates were molecularly identified using the genomic regions of internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) genes, and the sequences were deposited in GenBank (accession nos. QLYRR1, QLMCR9, QLMCR39: ON668272, ON668256, ON668257[ITS], ON684302, ON684300, ON684301[ACT], ON684316, ON684314, ON684315[CHS-1], ON684292, ON684290, ON684291[GAPDH], ON684286, ON684284, ON684285[TUB2]). The phylogenetic analysis of experimental strains was performed by Maximum-likelihood (ML) tree and Bayesian inference (BI) method. Nucleotide sequences exhibited three isolates were clustered with the ex-type strain C. pandanicola strain MFLUCC 170571T found in Thailand, C. pandanicola strains (SAUCC201152, SAUCC200204) found in Shandong Province, and the holotype stain C. parvisporum YMF 1.06942T found in Guangxi Province, China. Morphologically, isolates were easily distinguished from C. parvisporum by the colony on PDA and the size of conidia (Yu et al. 2022). Morphological characteristics and phylogenetic analyses revealed that QLYRR1, QLMCR9, and QLMCR39 belong to C. pandanicola, the members of the C. gloeosporioides species complex (Tibpromma et al. 2018; Mu et al. 2021). Koch's postulates were tested by strawberry plants (two cultivars, Akihime and Miaoxiang) in vivo, strawberry plants were tested for the three isolates by spraying 1×106 conidia/mL suspension on three seedlings. Three seedlings sprayed with sterile distilled water were served as control. All of the plants were transferred to a glasshouse with a 28/20 °C day/night temperature range and natural sunlight. After 6 weeks, QLYRR1-, QLMCR9-, and QLMCR39-sprayed seedlings were stunted and developed typical wilt symptoms similar to those observed in the field with the incidence for 3, 3, and 3 seedlings, respectively. The negative control remained asymptomatic. The fungi were reisolated again from lesions of diseased plants and leaves with 100% frequency, and morphological characteristics and tested gene sequences were identical to the original isolates in this note, thus fulfilling Koch's postulates. C. pandanicola was described from the healthy leaves of Pandanus sp. and the lesion fruits of Juglans regia. To our knowledge, this is the first report confirming C. pandanicola causes anthracnose crown rot on strawberries in China. C. pandanicola has the potential for causing serious losses to the strawberry industry, and research is needed on management strategies to minimize losses.

Endogenous bacteria inhabiting the Ophiocordyceps highlandensis during fruiting body development.

BACKGROUND: The genus Ophiocordyceps, which includes Ophiocordyceps sinensis, has been demonstrated to be one of the most valuable medicinal taxa. The low rate of larval infection and slow development that characterize the cultivation of this genus should be urgently addressed. To identify potential bioinoculants that stimulate the growth of Ophiocordyceps, O. highlandensis was selected as a model system, and a total of 72 samples were collected to systematically compare the microbial communities present during fruiting body development. By applying high-throughput 16S and ITS2 amplicon sequencing technology, the bacterial and fungal communities were identified in O. highlandensis and its surrounding soil, and the functional dynamics of the bacteria were explored. RESULTS: The results indicate that the most abundant bacteria across all the samples from O. highlandensis were Proteobacteria, Firmicutes and Bacteroidetes, while members of Ascomycota were detected among the fungi. The pathways enriched in the developmental stages were associated with carbohydrate degradation, nucleotides and pyridoxal biosynthesis, and the TCA cycle. Compared with that in the fungal community, an unexpectedly high taxonomic and functional fluctuation was discovered in the bacterial community during the maturation of O. highlandensis. Furthermore, bipartite network analysis identified four potential supercore OTUs associated with O. highlandensis growth. CONCLUSIONS: All the findings of this study suggest unexpectedly high taxonomic and functional fluctuations in the bacterial community of O. highlandensis during its maturation. O. highlandensis may recruit different endogenous bacteria across its life cycle to enhance growth and support rapid infection. These results may facilitate Ophiocordyceps cultivation and improve the development of strategies for the identification of potential bioinoculant resources.

Characterization of Novel Bacteriophage AhyVDH1 and Its Lytic Activity Against Aeromonas hydrophila.

Phage therapy is an alternative approach to overcome the problem of multidrug-resistant bacteria. Here, a novel bacteriophage AhyVDH1, which infects Aeromonas hydrophila 4572, was isolated and its morphology, one-step growth curve, lytic activity, stability under various conditions, and genome were investigated. Transmission electron microscopy revealed that AhyVDH1 has an icosahedral head 49 nm in diameter and a contractile tail 127 nm in length, suggesting that it belongs to the family Myoviridae. AhyVDH1 showed strong adsorption to the surface of A. hydrophila 4572 (90% in 10 min). The latent period of AhyVDH1 was shown to be 50 min, and the burst size was 274 plaque-forming unit/infected cell. AhyVDH1 was stable at 30 °C for 1 h and lost infectivity after20 min of heating at 60 °C. Infectivity remained unaffected at pH 6-7 for 1 h, while the bacteriophage was inactivated at pH < 4 or > 11. AhyVDH1 has a 39,175-bp genome, with a 58% G + C content and 59 open reading frames. BLAST analysis indicated that the genome sequence of phage AhyVDH1 was related to that of Aeromonas phage Ahp2. Both time and MOI-dependent in vitro A. hydrophila growth inhibition were observed with AhyVDH1.Re-growth of the host bacteria appeared about 12 h after treatment, suggesting its potential therapeutic value in treating A. hydrophila infections, but phage cocktails should be developed.

Comparison of the Bulk and Rhizosphere Soil Prokaryotic Communities Between Wild and Reintroduced Manglietiastrum sinicum Plants, a Threatened Species with Extremely Small Populations.

Huagaimu (Manglietiastrum sinicum) trees are critically endangered species and classified as a plant species with extremely small populations in China. Rhizospheres and bulk soils prokaryotic communities play an important role to protect and promote plants health and growth. However, the compositions and structures of prokaryotic communities in wild and reintroduced M. sinicum rhizospheres and bulk soils are still poorly understood. In the present study, prokaryotic communities in wild and reintroduced M. sinicum rhizospheres and bulk soils were compared using high-throughput sequencing. Thirty-two phyla, 76 classes, 193 orders, 296 families, and 470 genera of prokaryotes were obtained. Proteobacteria and Acidobacteria were the two most abundant phyla in all soil samples. The compositions and structures of prokaryotic communities were overall similar, and the abundance of some taxa varied significantly among soil samples. Soil prokaryotic communities were significantly affected by soil pH, total nitrogen, total phosphorus, and total potassium. Eleven of predicted functions were significantly different among the four soil groups. This study provides for the first insights into the compositions, structures, and potential functions of prokaryotic communities associated with wild and reintroduced M. sinicum rhizospheres and bulk soils, and providing a foundation for future research to help protect this endangered species.

DNA 5-Methylcytosine-Specific Amplification and Sequencing.

DNA 5-methylcytosine (5mC)-specific mapping has been hampered by severe DNA degradation and the presence of 5-hydroxymethylcytosine (5hmC) using the conventional bisulfite sequencing approach. Here, we present a 5mC-specific whole-genome amplification method (5mC-WGA), with which we achieved 5mC retention during DNA amplification from limited input down to 10 pg scale with limited interference from 5hmC signals, providing DNA 5mC methylome with high reproducibility and accuracy.

Lampropedia aestuarii sp. nov., isolated from the estuary of a freshwater river.

A Gram-stain-negative, strictly aerobic, catalase-positive and oxidase-positive bacterium, designated strain YIM MLB12T, was isolated from estuary sediment sampled at Maliao River where it flows into a plateau lake (Dianchi) in Yunnan, south-west PR China. Cells were non-motile and rod-shaped. Growth was observed at 15-35 °C (optimum, 25-30 °C), pH 6.0-10.0 (optimum, pH 7.0-8.0) and in the presence of 0-7 % (w/v) NaCl (optimum, 0.5-2 %). Results of phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM MLB12T formed a tight phylogenic lineage with members of the genus Lampropedia and was closely related to 'Lampropedia puyangensis' 2-bin with 98.3 % sequence similarity and had low similarities to the type strains of Lampropediahyalina ATCC 11041T (96 %) and Lampropedia cohaerens CT6T (95.5 %). Average nucleotide identity and in silico DNA-DNA hybridization values between strain YIM MLB12T and 'L. puyangensis' KCTC 32235 were 76.5 and 22.6 %, respectively. Strain YIM MLB12T contained ubiquinone-8 as the major quinone. The predominant cellular fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 0, C10 : 0 3-OH, summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c), C12 : 0 3-OH and C14 : 0. The polar lipid profile of strain YIM MLB12T was composed predominantly of diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The major polyamine was spermidine. The genomic DNA G+C content of strain YIM MLB12T was 56.8 mol%. Based on its genotypic and chemotaxonomic features and results of phenotypic analyses, strain YIM MLB12T represents a novel species of the genus Lampropedia, for which the name Lampropediaaestuarii sp. nov. is proposed. The type strain is YIM MLB12T (=KCTC 42886T=CGMCC 1.17071T).

Rhizospheric soil fungal community patterns of Duchesnea indica in response to altitude gradient in Yunnan, southwest China.

The magnitude of the impact of altitude gradient on microbial community and diversity has been studied in recent decades. Whereas bacteria have been the focus of most studies, fungi have been given relatively less attention. As a vital part of the macro- and microscopic ecosystem, rhizosphere fungi play a key role in organic matter decomposition and relative abundance of plant species and have an impact on plant growth and development. Using Duchesnea indica as the host plant, we examined the rhizosphere soil fungal community patterns across the altitude gradient in 15 sites of Yunnan province by sequencing the fungal ITS2 region with the Illumina MiSeq platform. We determined the fungal community composition and structure. We found that, surprisingly, rhizosphere soil fungal diversity of D. indica increased with altitudinal gradient. There was a slight difference in diversity between samples from high- and medium-altitude sites, with medium-altitude sites having the greater diversity. Furthermore, the rhizosphere soil fungal community composition and structure kept changing along the altitudinal gradient. Taxonomic results showed that the extent of phylum diversity was greatest at high-altitude sites, with Ascomycota, Basidiomycota, Zygomycota, and Glomeromycota as the most dominant fungal phyla.