Subgenome phasing for complex allopolyploidy: case-based benchmarking and recommendations.

Accurate subgenome phasing is crucial for understanding the origin, evolution and adaptive potential of polyploid genomes. SubPhaser and WGDI software are two common methodologies for subgenome phasing in allopolyploids, particularly in scenarios lacking known diploid progenitors. Triggered by a recent debate over the subgenomic origins of the cultivated octoploid strawberry, we examined four well-documented complex allopolyploidy cases as benchmarks, to evaluate and compare the accuracy of the two software. Our analysis demonstrates that the subgenomic structure phased by both software is in line with prior research, effectively tracing complex allopolyploid evolutionary trajectories despite the limitations of each software. Furthermore, using these validated methodologies, we revisited the controversial issue regarding the progenitors of the octoploid strawberry. The results of both methodologies reaffirm Fragaria vesca and Fragaria iinumae as progenitors of the octoploid strawberry. Finally, we propose recommendations for enhancing the accuracy of subgenome phasing in future studies, recognizing the potential of integrated tools for advanced complex allopolyploidy research and offering a new roadmap for robust subgenome-based phylogenetic analysis.

High-quality genome assembly enables prediction of allele-specific gene expression in hybrid poplar.

Poplar (Populus) is a well-established model system for tree genomics and molecular breeding, and hybrid poplar is widely used in forest plantations. However, distinguishing its diploid homologous chromosomes is difficult, complicating advanced functional studies on specific alleles. In this study, we applied a trio-binning design and PacBio high-fidelity long-read sequencing to obtain haplotype-phased telomere-to-telomere genome assemblies for the 2 parents of the well-studied F1 hybrid "84K" (Populus alba × Populus tremula var. glandulosa). Almost all chromosomes, including the telomeres and centromeres, were completely assembled for each haplotype subgenome apart from 2 small gaps on one chromosome. By incorporating information from these haplotype assemblies and extensive RNA-seq data, we analyzed gene expression patterns between the 2 subgenomes and alleles. Transcription bias at the subgenome level was not uncovered, but extensive-expression differences were detected between alleles. We developed machine-learning (ML) models to predict allele-specific expression (ASE) with high accuracy and identified underlying genome features most highly influencing ASE. One of our models with 15 predictor variables achieved 77% accuracy on the training set and 74% accuracy on the testing set. ML models identified gene body CHG methylation, sequence divergence, and transposon occupancy both upstream and downstream of alleles as important factors for ASE. Our haplotype-phased genome assemblies and ML strategy highlight an avenue for functional studies in Populus and provide additional tools for studying ASE and heterosis in hybrids.

Gap-free X and Y chromosome assemblies of Salix arbutifolia reveal an evolutionary change from male to female heterogamety in willows, without a change in the position of the sex-determining locus.

In the Vetrix clade of Salix, a genus of woody flowering plants, sex determination involves chromosome 15, but an XY system has changed to a ZW system. We studied the detailed genetic changes involved. We used genome sequencing, with chromosome conformation capture (Hi-C) and PacBio HiFi reads to assemble chromosome level gap-free X and Y of Salix arbutifolia, and distinguished the haplotypes in the 15X- and 15Y-linked regions, to study the evolutionary history of the sex-linked regions (SLRs). Our sequencing revealed heteromorphism of the X and Y haplotypes of the SLR, with the X-linked region being considerably larger than the corresponding Y region, mainly due to accumulated repetitive sequences and gene duplications. The phylogenies of single-copy orthogroups within the SLRs indicate that S. arbutifolia and Salix purpurea share an ancestral SLR within a repeat-rich region near the chromosome 15 centromere. During the change in heterogamety, the X-linked region changed to a W-linked one, while the Z was derived from the Y.

SubPhaser: a robust allopolyploid subgenome phasing method based on subgenome-specific k-mers.

With advanced sequencing technology, dozens of complex polyploid plant genomes have been characterized. However, for many polyploid species, their diploid ancestors are unknown or extinct, making it impossible to unravel the subgenomes and genome evolution directly. We developed a novel subgenome-phasing algorithm, SubPhaser, specifically designed for a neoallopolyploid or a homoploid hybrid. SubPhaser first searches for the subgenome-specific sequence (k-mer), then assigns homoeologous chromosomes into subgenomes, and further provides tools to annotate and investigate specific sequences. SubPhaser works well on neoallopolyploids and homoploid hybrids containing subgenome-specific sequences like wheat, but fails on autopolyploids lacking subgenome-specific sequences like alfalfa, indicating that SubPhaser can phase neoallopolyploid/homoploid hybrids with high accuracy, sensitivity and performance. This highly accurate, highly sensitive, ancestral data free chromosome phasing algorithm, SubPhaser, offers significant application value for subgenome phasing in neoallopolyploids and homoploid hybrids, and for the subsequent exploration of genome evolution and related genetic/epigenetic mechanisms.

Genome-wide analysis of butterfly bush (Buddleja alternifolia) in three uplands provides insights into biogeography, demography and speciation.

Understanding processes that generate and maintain large disjunctions within plant species can provide valuable insights into plant diversity and speciation. The butterfly bush Buddleja alternifolia has an unusual disjunct distribution, occurring in the Himalaya, Hengduan Mountains (HDM) and the Loess Plateau (LP) in China. We generated a high-quality, chromosome-level genome assembly of B. alternifolia, the first within the family Scrophulariaceae. Whole-genome re-sequencing data from 48 populations plus morphological and petal colour reflectance data covering its full distribution range were collected. Three distinct genetic lineages of B. alternifolia were uncovered, corresponding to Himalayan, HDM and LP populations, with the last also differentiated morphologically and phenologically, indicating occurrence of allopatric speciation likely to be facilitated by geographic isolation and divergent adaptation to distinct ecological niches. Moreover, speciation with gene flow between populations from either side of a mountain barrier could be under way within LP. The current disjunctions within B. alternifolia might result from vicariance of a once widespread distribution, followed by several past contraction and expansion events, possibly linked to climate fluctuations promoted by the Kunlun-Yellow river tectonic movement. Several adaptive genes are likely to be either uniformly or diversely selected among regions, providing a footprint of local adaptations. These findings provide new insights into plant biogeography, adaptation and different processes of allopatric speciation.

Sphingomonas floccifaciens sp. nov., isolated from subterranean sediment.

A Gram-stain-negative, non-motile, non-sporulating, rod-shaped, orange-pigmented bacterium, designated strain FQM01T, was isolated from a subterranean sediment sample in the Mohe permafrost area, China. Strain FQM01T grew optimally at 25 °C, pH 7.0 and NaCl concentration of 0 % (w/v). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain FQM01T belonged to the genus Sphingomonas. The closest phylogenetic relative was Sphingomonas spermidinifaciens GDMCC 1.657T (97.6 %), followed by Sphingomonas mucosissima DSM 17494T (97.2 %). The DNA G+C content of the isolate was 66.9 mol%. Strain FQM01T contained Q-10 as the predominant ubiquinone, and C18 : 1ω6c and/or C18 : 1ω7c, C16 : 1ω6c and/or C16 : 1ω7c, C16 : 0, C14 : 0 2-OH and C18 : 1ω7c 11 methyl as the major fatty acids. Major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and an unidentified glycolipid. Only sym-homospermidine was detected as the polyamine. On the basis of phylogenetic and phenotypic data, strain FQM01T is considered to represent a novel species of Sphingomonas for which the name Sphingomonasfloccifaciens sp. nov. is proposed. The type strain is FQM01T (=CGMCC 1.15797T=KCTC 52630T).

[Treatment of Unilateral Neglect using Repetitive Transcranial Magnetic Stimulation (rTMS) and Sensory Cueing (SC) in Stroke Patients].

OBJECTIVES: To evaluate the effectiveness of repetitive transcranial magnetic stimulation (rTMS) and sensory cueing (SC) for improving hemi-spatial attention deficits related to unilateral neglect, upper limb function and independence of stroke patients. METHODS: An assessor-blinded randomized controlled trial (RCT) was conducted. Eligible stroke patients were treated with rTMS (n =17) or rTMS combined with SC (n =16) in addition to conventional rehabilitation measures. rTMS was applied with low frequency (1 Hz) over the posterior parietal cortex (P5) of the lefthemisphere, 90% resting motor threshold, 900 pulses each session, one session per day, and 5 d per week for 2 weeks. SC was emitted using a wristwatch device attached to the hemiplegic arm for 2 weeks with a cumulative wear time of 3 h per day. The severity of unilateral neglect [behavioral inattention test conventional subtests (BITC), Catherine Bergego scale (CBS)], activity of daily living [modified Barthel index (MBI)], and upper limb function [Fugl-Meyer assessment (FMA), action research arm test (ARAT)] of the patients were measured pre- and post-interventions (immediately after 2 weeks' treatment) by an occupational therapist. RESULTS: BIT-C was relieved significantly over time in both groups. But rTMS+SC had greater improvement than rTMS alone (P <0.05). No significant differences was found between the two groups in other outcomes (CBS, FMA, ARAT). CONCLUSION: rTMS combined with SC is better than rTMS alone for treating unilateral neglect in stroke patients.

Description of Sphingomonas mohensis sp. nov., Isolated from Sediment.

An aerobic, gram-reaction-negative, rod-shaped bacterium, designated strain Z6(T), was isolated from sediment collected at Mohe Basin, China. And its taxonomic position was investigated by applying a polyphasic approach. Growth occurs at 10-45 °C (optimum, 30 °C), at pH 6.0-11.0 (optimum, pH 7.0) and in the presence of 0-3.0 % (w/v) NaCl (optimum, 0 %). The polar lipid profile of strain Z6(T) revealed the presence of diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid and two unidentified phospholipids, and the major quinone was Q-10. The major fatty acids were C18:1 ω7c and/or C18:1 ω6c (summed feature 8) and C16:1 ω6c and/or C16:1 ω7c (summed feature 3). The predominant polyamine was homospermidine. The DNA G + C content of strain Z6(T) is 65.2 mol%. On the basis of the polyphasic evidence presented, strain Z6(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas mohensis sp. nov. is proposed. The type strain is Z6(T) (=CGMCC 1.12891(T) = JCM 19983(T)).

Dyadobacter sediminis sp. nov., isolated from a subterranean sediment sample.

A Gram-reaction-negative, flexirubin-type-pigmented, rod-shaped, aerobic, non-motile bacterium, designated strain Z12(T), was isolated from a subsurface sediment sample. In a phylogenetic tree based on 16S rRNA gene sequences, strain Z12(T) formed a distinct clade with the members of the genus Dyadobacter (<96.7 % sequence similarity). The G+C content of genomic DNA was 45.4 %. The major fatty acids of strain Z12(T) were iso-C15 : 0, C16 : 1ω6c and/or C16 : 1ω7c (summed feature 3) and anteiso-C17 : 1 B and/or iso-C17 : 1 I (summed feature 4). The major respiratory quinone was MK-7 and the major polar lipid was phosphatidylethanolamine. On the basis of phenotypic, phylogenetic and genotypic features, strain Z12(T) is considered to represent a novel species, for which the name Dyadobacter sediminis sp. nov., is proposed. The type strain is Z12(T) ( = JCM 30073(T) = CGMCC 1.12895(T)).

Arenimonas maotaiensis sp. nov., isolated from fresh water.

A translucent, white, Gram-reaction-negative, facultatively anaerobic, non-flagellated, slightly curved or curved bacterial strain, designated YT8(T), was isolated from the fresh water of the Maotai section of Chishui River, China. Cells were catalase-positive and oxidase-positive. Phylogenetic analyses of 16S rRNA gene sequences revealed that strain YT8(T) is a member of the genus Arenimonas with similarity to other members of this genus ranging from 93.7 to 95.0 %. The major isoprenoid quinone was ubiquinone 8 (Q-8), major polar lipids were phosphatidylethanolamine, one unidentified aminolipid, two unidentified phospholipids and two unidentified polar lipids, while major fatty acids were iso-C15 : 0, iso-C14 : 0 and anteiso-C15 : 0. The DNA G+C content of strain YT8(T) was 66.6 mol%. On the basis of phenotypic, phylogenetic and genotypic features studied, strain YT8(T) is suggested to represent a novel species of the genus Arenimonas, for which the name Arenimonas maotaiensis sp. nov. is proposed. The type strain is YT8(T) ( = CGMCC 1.12726(T) = JCM 19710(T)).

Moheibacter sediminis gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from sediment, and emended descriptions of Empedobacter brevis, Wautersiella falsenii and Weeksella virosa.

A Gram-reaction-negative, yellow-pigmented, strictly aerobic bacterium, designated M0116T, was isolated from the sediment of the Mohe Basin in north-east China. Flexirubin-type pigments were produced. Cells were catalase- and oxidase-positive and non-gliding rods. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain M0116T was a member of the family Flavobacteriaceae and was most closely related to members of the genera Empedobacter, Wautersiella and Weeksella with 90.5-91.0% sequence similarities. The major cellular fatty acids were iso-C15:0 and iso-C17:0 3-OH. The major respiratory quinone was MK-6 and the major polar lipid was phosphatidylethanolamine. The DNA G+C content was 38.2 mol%. Based on phenotypic, phylogenetic and genotypic data, strain M0116T is considered to represent a novel species of a new genus in the family Flavobacteriaceae, for which the name Moheibacter sediminis gen. nov., sp. nov. is proposed. The type strain is M0116T (=CGMCC 1.12708T=JCM 19634T). Emended descriptions of Empedobacter brevis, Wautersiella falsenii and Weeksella virosa are also proposed.

Description of Chishuiella changwenlii gen. nov., sp. nov., isolated from freshwater, and transfer of Wautersiella falsenii to the genus Empedobacter as Empedobacter falsenii comb. nov.

A Gram-reaction-negative, strictly aerobic, non-pigmented, non-gliding, rod-shaped bacterium, designated strain BY4(T), was isolated from freshwater. Cells were catalase- and oxidase-positive and indole was produced. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain BY4(T) belonged to the family Flavobacteriaceae and showed 91.6-95.9% sequence similarities to the most closely related strains. The major respiratory quinone was MK-6 and the major polar lipid was phosphatidylethanolamine. The major polyamine was homospermidine and the major fatty acids were iso-C(15 : 0), iso-C(17 : 0) 3-OH and summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c). The DNA G+C content was 30.0 mol%. On the basis of phenotypic, phylogenetic and genotypic features, strain BY4(T) is suggested to represent a novel species in a new genus within the family Flavobacteriaceae, for which the name Chishuiella changwenlii gen. nov., sp. nov. is proposed. The type strain of this type species is BY4(T) ( = CGMCC 1.12707(T) = JCM 19633(T)). On the basis of data collected from previous and present studies, it is proposed to reclassify Wautersiella falsenii to the genus Empedobacter as the new combination Empedobacter falsenii comb. nov. (type strain NF 993(T) = CCUG 51536(T) = CIP 108861(T)).

Taibaiella chishuiensis sp. nov., isolated from freshwater.

A Gram-reaction-negative, strictly aerobic, non-motile and rod-shaped bacterial strain, AY17T, was isolated from the Chishui River in Guizhou Province, South-west China. Strain AY17T grew optimally at pH 7.0 and 20 °C. Flexirubin-type pigments were produced. 16S rRNA gene sequence analysis indicated that strain AY17T belonged to the family Chitinophagaceae within the phylum Bacteroidetes; the closest phylogenetic relative was Taibaiella smilacinae PTJT-5T (95.3% gene sequence similarity). The DNA G+C content was 49.5 mol%. Strain AY17T contained MK-7 as the predominant respiratory quinone and phosphatidylethanolamine as the major polar lipid. The major fatty acids were iso-C15:0, iso-C15:1G and iso-C17:0 3-OH. On the basis of phylogenetic, phenotypic and genetic data, strain AY17T was classified as representing a novel species of the genus Taibaiella, for which the name Taibaiella chishuiensis sp. nov. is proposed. The type strain is AY17T (=CGMCC 1.12700T=JCM 19637T).

[Molecular cloning and localization of Leishmania donovani expression site associated genes-like protein].

OBJECTIVE: To clone the novel gene that specifically expressed in the amastigotes of Leishmania donovani, and observe subcellular localization of the gene encoding protein. METHODS: mRNA from promastigotes and amastigotes of L. donovani were prepared. The novel expressed sequence tag of amastigotes was selected by suppression subtractive hybridization. The expression of the novel gene in different stages of L. donovani was detected by Northern hybridization and semi-quantitative RT-PCR. The subcellular localization of the novel gene encoding protein was observed. RESULTS: The subtractive library of the specifically expressed sequence tag of amastigotes was constructed, and a novel gene designated as expression site associated genes-like protein (ESAGLP) gene was cloned. The full length of ESAGLP cDNA was 2,258 bp. The open-reading frame encoded a polypeptide of 620 amino acid residues. ESAGLP gene expressed only in amastigotes, the encoding protein was localized in the mitochondria. CONCLUSION: The ESAGLP gene is identified as a novel gene which specifically expressed in Leishmania donovani amastigotes, and its encoding protein is localized in the mitochondria.

Unravelling the novel sex determination genotype with 'ZY' and a distinctive 2.15-2.95 Mb inversion among poplar species through haplotype-resolved genome assembly and comparative genomics analysis.

Populus tomentosa, an indigenous tree species, is widely distributed and cultivated over 1,000,000 km2 in China, contributing significantly to forest production, ecological conservation and urban-rural greening. Although a reference genome is available for P. tomentosa, the intricate interspecific hybrid origins, chromosome structural variations (SVs) and sex determination mechanisms remain confusion and unclear due to its broad and even overlapping geographical distribution, extensive morphological variations and cross infiltration among white poplar species. We conducted a haplotype-resolved de novo assembly of P. tomentosa elite individual GM107, which comprises subgenomes a and b with a total genome size of 714.9 Mb. We then analysed the formation of hybrid species and the phylogenetic evolution and sex differentiation across the entire genus. Phylogenomic analyses suggested that GM107 likely originated from a hybridisation event between P. alba (♀) and P. davidiana (♂) approximately 3.8 Mya. A total of 1551 chromosome SVs were identified between the two subgenomes. More noteworthily, a distinctive inversion structure spanning 2.15-2.95 Mb was unveiled among Populus, Tacamahaca, Turaga, Aigeiros poplar species and Salix, highlighting a unique evolutionary feature. Intriguingly, a novel sex genotype of the ZY type, which represents a crossover between XY and ZW systems, was identified and confirmed through both natural and artificial hybrids populations. These novel insights offer significant theoretical value for the study of the species' evolutionary origins and serve as a valuable resource for ecological genetics and forest biotechnology.

Haplotype-resolved genome assembly of Coriaria nepalensis a non-legume nitrogen-fixing shrub.

Coriaria nepalensis Wall. (Coriariaceae) is a nitrogen-fixing shrub which forms root nodules with the actinomycete Frankia. Oils and extracts of C. nepalensis have been reported to be bacteriostatic and insecticidal, and C. nepalensis bark provides a valuable tannin resource. Here, by combining PacBio HiFi sequencing and Hi-C scaffolding techniques, we generated a haplotype-resolved chromosome-scale genome assembly for C. nepalensis. This genome assembly is approximately 620 Mb in size with a contig N50 of 11 Mb, with 99.9% of the total assembled sequences anchored to 40 pseudochromosomes. We predicted 60,862 protein-coding genes of which 99.5% were annotated from databases. We further identified 939 tRNAs, 7,297 rRNAs, and 982 ncRNAs. The chromosome-scale genome of C. nepalensis is expected to be a significant resource for understanding the genetic basis of root nodulation with Frankia, toxicity, and tannin biosynthesis.

Telomere-to-telomere and haplotype-resolved genome assembly of the Chinese cork oak (Quercus variabilis).

The Quercus variabilis, a deciduous broadleaved tree species, holds significant ecological and economical value. While a chromosome-level genome for this species has been made available, it remains riddled with unanchored sequences and gaps. In this study, we present a nearly complete comprehensive telomere-to-telomere (T2T) and haplotype-resolved reference genome for Q. variabilis. This was achieved through the integration of ONT ultra-long reads, PacBio HiFi long reads, and Hi-C data. The resultant two haplotype genomes measure 789 Mb and 768 Mb in length, with a contig N50 of 65 Mb and 56 Mb, and were anchored to 12 allelic chromosomes. Within this T2T haplotype-resolved assembly, we predicted 36,830 and 36,370 protein-coding genes, with 95.9% and 96.0% functional annotation for each haplotype genome. The availability of the T2T and haplotype-resolved reference genome lays a solid foundation, not only for illustrating genome structure and functional genomics studies but also to inform and facilitate genetic breeding and improvement of cultivated Quercus species.

Gapless genome assembly of azalea and multi-omics investigation into divergence between two species with distinct flower color.

The genus Rhododendron (Ericaceae), with more than 1000 species highly diverse in flower color, is providing distinct ornamental values and a model system for flower color studies. Here, we investigated the divergence between two parental species with different flower color widely used for azalea breeding. Gapless genome assembly was generated for the yellow-flowered azalea, Rhododendron molle. Comparative genomics found recent proliferation of long terminal repeat retrotransposons (LTR-RTs), especially Gypsy, has resulted in a 125 Mb (19%) genome size increase in species-specific regions, and a significant amount of dispersed gene duplicates (13 402) and pseudogenes (17 437). Metabolomic assessment revealed that yellow flower coloration is attributed to the dynamic changes of carotenoids/flavonols biosynthesis and chlorophyll degradation. Time-ordered gene co-expression networks (TO-GCNs) and the comparison confirmed the metabolome and uncovered the specific gene regulatory changes underpinning the distinct flower pigmentation. B3 and ERF TFs were found dominating the gene regulation of carotenoids/flavonols characterized pigmentation in R. molle, while WRKY, ERF, WD40, C2H2, and NAC TFs collectively regulated the anthocyanins characterized pigmentation in the red-flowered R simsii. This study employed a multi-omics strategy in disentangling the complex divergence between two important azaleas and provided references for further functional genetics and molecular breeding.

Deferoxamine/magnesium modified ß-tricalcium phosphate promotes the bone regeneration in osteoporotic rats.

Recently, Deferoxamine (DFO) and magnesium (Mg) have been identified as critical factors for angiogenesis and bone formation. However, in current research studies, there is a lack of focus on whether DFO plus Mg can affect the regeneration of ß-tricalcium phosphate (ß-TCP) in osteoporosis and through what biological mechanisms. Therefore, the present work was aimed to preparation and evaluate the effect of Deferoxamine/magnesium modified ß-tricalcium phosphate promotes (DFO/Mg-TCP) in ovariectomized rats model and preliminary exploration of possible mechanisms. The MC3T3-E1 cells were co-cultured with the exudate of DFO/Mg-TCP and induced to osteogenesis, and the cell viability, osteogenic activity were observed by Cell Counting Kit-8(CCK-8), Alkaline Phosphatase (ALP) staining, Alizarin Red Staining (RES) and Western Blot. In vitro experiments, CCK-8, ALP and ARS staining results show that the mineralization and osteogenic activity of MC3T3-E1increased significantly after intervention by DFO/Mg-TCP, as well as a higher levels of protein expressions including VEGF, OC, Runx-2 and HIF-1α. In vivo experiment, Micro-CT and Histological analysis evaluation show that DFO/Mg-TCP treatment presented the stronger effect on bone regeneration, bone mineralization and biomaterial degradation, when compared with OVX+Mg-TCP group and OVX+TCP group, as well as a higher VEGF, OC, Runx-2 and HIF-1α gene expression. The present study indicates that treatment with DFO/Mg-TCP was associated with increased regeneration by enhancing the function of osteoblasts in an OVX rat.