Application of Functional Markers to Identify Genes for Bacterial Blight Resistance in Oryza rufipogon

Field resistances of nine accessions of common wild rice （Oryza rufipogon Griff.） and one rice variety （IR24） were evaluated by using nine strains of bacterial blight pathogen （Xanthomonas oryzae pv. oryzae） from the Philippines. IR24 was highly susceptible to all the strains, and six common wild rice accessions resisted all the nine strains, with a resistance frequency of 67%. The accessions Yulin and Wanning were only susceptible to PXO280 and PXO71, respectively. The accession Gaozhou was susceptible to the three strains PXO79, PXO99 and PXO339, whereas resistant to the other six strains. It could be concluded that there is at least one resistance gene in each common wild rice accession. The functional markers of the genes xa5, xa13, Xa21 and Xa27 were used to detect the presence of these resistance genes in the nine tested wild rice accessions, and it was found that four wild rice accessions contained heterozygous xa13. Among the nine common wild rice accessions, five were homozygous for Xa27 and three homozygous for xa27, and the accession Laibin contained neither xa27 nor Xa27. In addition, there were no xa5 and Xa21 in all of these accessions.

Construction of Expression Vector Capable of Excising Selectable Marker Gene

[ Objective] This study was to construct an expression vector capable of excising selectable marker gene, further eliminating the effect of marker gene on the functional study of target gene. [ Method] By using Cre/LoxP site-specific recombination system, DsRed2-1 vector was modified by introducing LoxP se- quence and multiple cloning site sequence, then the TK gene was ligated into this vector for negative selection. [ Results] The fragments introduced were recom- bined at the molecular level under induced condition, and the specific red fluorescence was also observed in the recombinant vector transfected cells at the cellular level. [ Conclusion] It is feasible to use this Cre/loxP system to excise marker genes, showing a broad application prospect.

Postulation of seedling leaf rust resistance genes in 84 Chinese winter wheat cultivars

Wheat leaf rust（caused by Puccinia triticina） is one of the most important fungal diseases in China. There are tens of winter wheat cultivars which are approved to be released by the government at a national level and more than 100 wheat cultivars at the provincial level. But there is no information about leaf rust（Lr） genes in these cultivars, which makes it difficult for farmers and breeders to select which cultivars they should plant in their fields and use in their breeding programs. The objective of this paper was to identify the leaf rust resistant genes at seedling stage present in the 84 commercial wheat cultivars from China that have been released in the past few years. A set of 20 near isogenic lines with Thatcher background and 6 lines with known Lr genes were used to test the virulence of 12 races of P. triticina（Pt）. By comparing the infection types（ITs） produced on the 84 cultivars by the 12 Pt races with the ITs on the differential sets, the Lr genes were postulated. In addition, 8 molecular markers of Lr genes such as Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr26 and Lr29, which are closely linked to or co-segregated with the Lr gene, were used for further validation of the genes in the 84 Chinese winter wheat cultivars. Twelve Lr genes, including Lr1, Lr3,（Lr3bg）,（Lr3ka）, Lr11, Lr13, Lr14 a, Lr16, Lr26, Lr27, Lr30 and Lr31 were postulated to be present either singly or in combinations in these Chinese wheat cultivars. Lr3 and Lr26 were detected most often in the tested cultivars, with frequencies of 51.2 and 38.1%, respectively. No wheat Lr genes were detected in 16 cultivars, and 4 cultivars may carry unknown Lr genes other than those used in this study. Lr9, Lr20, Lr21, Lr24, Lr25 and Lr29 were not present in any of the 84 tested accessions.

AFLP Marker Linked to Turnip Mosaic Virus Susceptible Gene in Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Turnip mosaic virus (TuMV) which has several strains causes the most important virusdisease in Chinese cabbage in terms of crop damage. In China, Chinese cabbage is infectedby a mixture of strains, breeding of cultivar for the TuMV resistance has become themajor aim. Screening the molecular marker linked to the TuMV-resistance gene formolecular assisted selection is the major method to improve the breeding efficiency. Inthis study, we used AFLP technique and the method of bulked segregant analysis(BSA) tostudy the progeny of Brp0058Brp0108, and identified two DNA molecular marker linked toTurnip mosaic virus-resistance gene with a recombination frequency 7.5 cM and 8.4 cM.

Identification of Molecular Markers Linked to the Wilt Resistance Gene FuJ7(t) in Flax

A cross between wilt resistant flax variety Jinya7 and susceptible variety Jinya1 wasmade for mapping wilt resistance gene(s). The inoculation test of F1 and F2 progeny provedthat the resistance of Jinya7 to wilt is controlled by two dominant genes. With 48 EcoRⅠ/MseⅠ primer combinations, amplified fragment length polymorphisms (AFLP) analysis wasperformed on two parents and their F2 resistance and susceptibility bulks. A total ofabout 3300 distinguishable bands were amplified, of which three bands had stabledifferences. The genetic linkage analysis of the three polymorphic DNA fragments withthe resistance gene(s) was made in the F2 segregating population derived from the crossbetween Jinya7 and Jinya1. The DNA fragment AG/CAG was found closely linked to one of thewilt-resistant genes, which with a genetic distance of 5.2cm, was tentatively named FuJ7(t).The cloned fragment AG/CAG was sequenced and then converted successfully to a sequencecharacterized amplified region (SCAR) marker, which can be used more conveniently in theidentification and marker-assisted selection for the wilt resistance gene FuJ7(t) toflax wilt.

Identification of a RAPD marker linked to a blast resistance gene in Oryza sativa L

Marker-aided selection has received more attention in recent years. This relies on the exploitation of dose linkage between molecular markers and target gene(s). We report here a randomly amplified polymorphic DNA (RAPD) marker tightly linked to the blast resistance gene Pi-ll(t) derived from Hongjiaozhan, which confers the resistance to race ZB1 of Pyricularia oryzae Cav.

Mapping of putative sucking inhibitory gene to whitebacked planthopper by linkage analysis with CAPS markers

Resistance to whitebacked planthopper(WBPH)in Chinese japonica riceChunjiang 06(CJ-06)was mediated by sucking inhibitory and ovicidal mecha-nism.The ovicidal reponse was a common self-defense mechanism againstWBPH in japonica.The ovicidal gene and its chromosomal position had alreadybeen identified.The sucking inhibitory nature of CJ-06 caused a definenon-preference behavior of WBPH in fields.A single dominant gene governed

Diversity,community structure,and quantity of eukaryotic phytoplankton revealed using 18S rRNA and plastid 16S rRNA genes and pigment markers:a case study of the Pearl River Estuary

Understanding consistencies and discrepancies in characterizing diversity and quantity of phytoplankton is essential for better modeling ecosystem change.In this study,eukaryotic phytoplankton in the Pearl River Estuary,South China Sea were investigated using nuclear 18S rRNA and plastid 16S or 23S rRNA genes and pigment analysis.It was found that 18S abundance poorly explained the variations in total chlorophyll a(Chl-a).However,the ratios of log-transformed 18S abundance to Chl-a in the major phytoplankton groups were generally environment dependent,suggesting that the ratio has potential as an indicator of the physiological state of phytoplankton.The richness of 18S-based operational taxonomic units was positively correlated with the richness of 16S-based amplicon sequence variants of the whole phytoplankton community,but insignifcant or weak for individual phytoplankton groups.Overall,the 18S based,rather than the 16S based,community structure had a greater similarity to pigment-based estimations.Relative to the pigment data,the proportion of haptophytes in the 18S dataset,and diatoms and cryptophytes in the 16S dataset,were underestimated.This study highlights that 18S metabarcoding tends to refect biomass-based community organization of eukaryotic phytoplankton.Because there were lower copy numbers of plastid 16S than 18S per genome,metabarcoding of 16S probably approximates cell abundance-based community organization.Changes in biomass organization of the pigment-based community were sensitive to environmental changes.Taken together,multiple methodologies are recommended to be applied to more accurately profle the diversity and community composition of phytoplankton in natural ecosystems.

Single-cell and spatial heterogeneity landscapes of mature epicardial cells

Tbx18,Wt1,and Tcf21 have been identified as epicardial markers during the early embryonic stage.However,the gene markers of mature epicardial cells remain unclear.Single-cell transcriptomic analysis was performed with the Seurat,Monocle,and CellphoneDB packages in R software with standard procedures.Spatial transcriptomics was performed on chilled Visium Tissue Optimization Slides(10x Genomics)and Visium Spatial Gene Expression Slides(10x Genomics).Spatial transcriptomics analysis was performed with Space Ranger software and R software.Immunofluorescence,whole-mount RNA in situ hybridization and X-gal staining were performed to validate the analysis results.Spatial transcriptomics analysis revealed distinct transcriptional profiles and functions between epicardial tissue and non-epicardial tissue.Several gene markers specific to postnatal epicardial tissue were identified,including Msln,C3,Efemp1,and Upk3b.Single-cell transcriptomic analysis revealed that cardiac cells from wildtype mouse hearts(from embryonic day 9.5 to postnatal day 9)could be categorized into six major cell types,which included epicardial cells.Throughout epicardial development,Wt1,Tbx18,and Upk3b were consistently expressed,whereas genes including Msln,C3,and Efemp1 exhibited increased expression during the mature stages of development.Pseudotime analysis further revealed two epicardial cell fates during maturation.Moreover,Upk3b,Msln,Efemp1,and C3 positive epicardial cells were enriched in extracellular matrix signaling.Our results suggested Upk3b,Efemp1,Msln,C3,and other genes were mature epicardium markers.Extracellular matrix signaling was found to play a critical role in the mature epicardium,thus suggesting potential therapeutic targets for heart regeneration in future clinical practice.

Single-cell transcriptome analysis reveals a cancer-associated fibroblast marker gene signature in hepatocellular carcinoma that predicts prognosis

Background and aims:Hepatocellular carcinoma(HCC)is one of the leading causes of cancer death.Multi-pathway combination therapy is used to treat HCC,and immunotherapy is also a routine part of treatment.As a major component of the tumor microenvironment(TME),cancer-associated fibroblasts(CAFs)actively participate in cancer progression through complex functions.However,because CAFs dynamically change during cancer development,most of the current treatment strategies targeting CAFs fail.We created a prognostic CAF marker gene signature(CAFMGS)to investigate the utility of CAFs as a prognostic factor and therapeutic target.Methods:Gene Expression Omnibus(GEO)single-cell RNA sequencing(Sc-RNA-seq)data were analyzed to identify CAF marker genes in HCC.The Cancer Genome Atlas(TCGA)database was used as a training cohort to construct the CAFMGS model and the International Cancer Genome Consortium(ICGC)dataset was used to validate the CAFMGS.Results:Marker genes in the CAFMGS model were(0.0001-SPP1),(0.0084-VCX3A),(0.0015-HMGA1),(0.0082-PLOD2),and(0.0075-CACYBP).The CAFMGS_score was separated into high-risk and low-risk groups based on the median of the patients’OS.Univariate and multivariate analyses confirmed that CAFMGS_score was an independent prognostic factor in the training group.CAFMGS_score was a more accurate prognostic indicator compared with clinicopathological score and tumor mutational burden score.Conclusion:CAFMGS offers a fresh perspective on stromal cell marker genes in HCC prognosis and expands our knowledge of CAF heterogeneity and functional diversity,perhaps paving the way for CAF-targeted immunotherapy in HCC patients.

Survival of Pseudomonas fluorescence X16(luxAB)Strain in Soils Accumulated with Mixed Rare Earth Elements

Rare earth elements(REE)are applied as micro-fertilizer in large scale in China and there is growing concern about the environmental effects of REE accumulation in soils. Accumulation of REE was simulated in lab by adding REE to three soils and the survival of Pseudomonas fluorescence X16 strain marked with luxAB gene in soils was detected. Curvilinear regression method was applied to analyze the survival pattern. The stimulation values, EC_(50) and NOEC values for X16 strain were calculated to compare the toxic intensity of REE in different soils. The stimulation(peak)values in red soil, yellow fluovo-aquic soil and yellow cinnamon soil, are 11.55～18.08,(0～2.13), 2.37～4.62 mg·kg^(-1) , respectively. EC_(50) values are 13.47～39.12, 6.59～56.18, 372～1034 (mg·kg^(-1)), respectively.NOEC values are 5.62 ～21.41, 0.00～4.53, 133.3～327.1 mg·kg^(-1), respectively. Tangents values of regression equation of the survival of X16 strain in red soil are the maximum ones indicating that REE accumulation in red soil has stronger inhibitory effects than in other two soils. The soil order, reflecting toxic intensity of REE is as follows: red soil>yellow fluovic-aquic soil>yellow cinnamon soil.

Profile of biological characterizations and clinical application of corneal stem/progenitor cells

Corneal stem/progenitor cells are typical adult stem/progenitor cells.The human cornea covers the front of the eyeball,which protects the eye from the outside environment while allowing vision.The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role.Corneal stem/progenitor cells include mainly corneal epithelial stem cells,corneal endothelial cell progenitors and corneal stromal stem cells.Since the discovery of corneal epithelial stem cells(also known as limbal stem cells)in 1971,an increasing number of markers for corneal stem/progenitor cells have been proposed,but there is no consensus regarding the definitive markers for them.Therefore,the identification,isolation and cultivation of these cells remain challenging without a unified approach.In this review,we systematically introduce the profile of biological characterizations,such as anatomy,characteristics,isolation,cultivation and molecular markers,and clinical applications of the three categories of corneal stem/progenitor cells.

Molecular Mapping of a Stripe Rust Resistance Gene YrH9020a Transferred from Psathyrostachys huashanica Keng on Wheat Chromosome 6D

Stripe rust （yellow rust）, caused by Puccinia striiformis f. sp. tritici （Pst）, is one of the most devastating diseases of wheat throughout the world. H9020-1-6-8-3 is a translocation line originally developed from interspeciifc hybridization between wheat line 7182 and Psathyrostachys huashanica Keng and is resistant to most Pst races in China. To identify the resistance gene（s） in the translocation line, H9020-1-6-8-3 was crossed with susceptible cultivar Mingxian 169, and seedlings of the parents, F1, F2, F3, and BC1 generations were tested with prevalent Chinese Pst race CYR32 under controlled greenhouse conditions. The results indicated that there is a single dominant gene, temporarily designated as YrH9020a, conferring resistance to CYR32. The resistance gene was mapped by the F2 population from Mingxian 169/H9020-1-6-8-3. It was linked to six microsatellite markers, including Xbarc196, Xbarc202, Xbarc96, Xgpw4372, Xbarc21, and Xgdm141, lfanked by Xbarc96 and Xbarc202 with at 4.5 and 8.3 cM, respectively. Based on the chromosomal locations of these markers and the test of Chinese Spring （CS） nullitetrasomic and ditelosomic lines, the gene was assigned to chromosome 6D. According to the origin and the chromosomal location, YrH9020a might be a new resistance gene to stripe rust. The lfanking markers linked to YrH9020a could be useful for marker-assisted selection in breeding programs.

A Modified Ant Colony Optimization Algorithm for Tumor Marker Gene Selection

Microarray data are often extremely asymmetric in dimensionality, such as thousands or even tens of thousands of genes but only a few hundreds of samples or less. Such extreme asymmetry between the dimensionality of genes and samples can lead to inaccurate diagnosis of disease in clinic. Therefore, it has been shown that selecting a small set of marker genes can lead to improved classification accuracy. In this paper, a simple modified ant colony optimization （ACO） algorithm is proposed to select tumorelated marker genes, and support vector machine （SVM） is used as classifier to evaluate the performance of the extracted gene subset. Experimental results on several benchmark tumor microarray datasets showed that the proposed approach produces better recognition with fewer marker genes than many other methods. It has been demonstrated that the modified ACO is a useful tool for selecting marker genes and mining high dimension data

Verification of Bacteroidales 16S rRNA markers as a complementary tool for detecting swine fecal pollution in the Yangtze Delta

To correctly assess and properly manage the public health risks associated with exposure to contaminated water,it is necessary to identify the source of fecal pollution in a watershed.In this study,we evaluated the efficacy of our two previously developed real time-quantitative PCR(qPCR)assays for the detection of swine-associated Bacteroidales genetic markers(gene 1-38,gene 3-53)in the Yangtze Delta watershed of southeastern China.The results indicated that the gene 1-38 and 3-53 markers exhibited high accuracy(92.5%,91.7%conditional probability,respectively)in detecting Bacteroidales spp.in water samples.According to binary logistic regression(BLR),these two swine-associated markers were well correlated(P<0.05)with fecal indicators(Escherichia coli and Enterococci spp.)and zoonotic pathogens(E.coli O157:H7,Salmonella spp.and Campylobacter spp.)in water samples.In contrast,concentrations of conventional fecal indicator bacteria(FIB)were not correlated with zoonotic pathogens,suggesting that they are noneffective at detecting fecal pollution events.Collectively,the results obtained in this study demonstrated that a swinetargeted qPCR assay based on two Bacteroidales genes markers(gene 1-38,gene 3-53)could be a useful tool in determining the swine-associated impacts of fecal contamination in a watershed.

Glow in the Dark： Fluorescent Proteins as Cell and Tissue-Specific Markers in Plants

Since the hallmark discovery of Aequorea victoria＇s Green Fluorescent Protein （GFP） and its adaptation for efficient use in plants, fluorescent protein tags marking expression profiles or genuine proteins of interest have been used to recognize plant tissues and cell types, to monitor dynamic cell fate selection processes, and to obtain cell type-specific transcriptomes. Fluorescent tagging enabled visualization in living tissues and the precise recordings of dy- namic expression pattern changes. The resulting accurate recording of cell fate acquisition kinetics in space and time has strongly stimulated mathematical modeling of self-organizing feedback mechanisms. In developmental studies, the use of fluorescent proteins has become critical, where morphological markers of tissues, cell types, or differentiation stages are either not known or not easily recognizable. In this review, we focus on the use of fluorescent markers to identify and illuminate otherwise invisible cell states in plant development.

A practical guide to amplicon and metagenomic analysis of microbiome data

Advances in high-throughput sequencing(HTS)have fostered rapid developments in the field of microbiome research,and massive microbiome datasets are now being generated.However,the diversity of software tools and the complexity of analysis pipelines make it difficult to access this field.Here,we systematically summarize the advantages and limitations of microbiome methods.Then,we recommend specific pipelines for amplicon and metagenomic analyses,and describe commonly-used software and databases,to help researchers select the appropriate tools.Furthermore,we introduce statistical and visualization methods suitable for microbiome analysis,including alpha-and betadiversity,taxonomic composition,difference comparisons,correlation,networks,machine learning,evolution,source tracing,and common visualization styles to help researchers make informed choices.Finally,a stepby-step reproducible analysis guide is introduced.We hope this review will allow researchers to carry out data analysis more effectively and to quickly select the appropriate tools in order to efficiently mine the biological significance behind the data.

Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus

High-throughputsingle-cellRNAsequencing(sc RNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba × Populus glandulosa. The sc RNA-seq profiled9,798 cells, which were grouped into 12 clusters.Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations,we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type(cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.

Development of islet organoids from human induced pluripotent stem cells in a cross-linked collagen scaffold

Islets organoids would have value in the cell replacement therapy for diabetes apart from usual personalized drug screening routes.Generation of a large number of Islets like clusters,with ability to respond to glucose stimulation appears to be an ideal choice.In this study we have generated islet organoids with the ability to respond to glucose stimulation by insulin release.The source of the cells was an iPSC cell line differentiated into the pancreatic progenitors.These cells were assembled in matrigel or cross-linked collagen scaffold and compared for their efficacy to release insulin upon stimulation with glucose.The assembled organoids were examined by immunohistochemistry and expression of the relevant marker genes.The organoids showed expression of islet like markers in both-matrigel and crosslinked collagen scaffold.The islet organoids in both the cases showed release of insulin upon stimulation with glucose.The crosslinked collagen scaffold is quite stable and supports islet cells growth and function.

Rescue of white egg 1 mutant by introduction of the wildtype Bombyx kynurenine 3-monooxygenase gene

In silkworms, the white egg 1 （w-1） mutant, which is characterized by white eyes and white eggs, is deficient in Bombyx kynurenine 3-monooxygenase （KMO） activity. To investigate whether the w-1 mutant phenotype is rescued by introducing the wild-type KMO gene, we constructed transgenic silkworms with the wild-type Bombyx KMO gene under the control of either the cytoplasmic actin gene promoter （A3KMO） or the native KMO gene promoter （KKMO）. We created two transgenic lines with A3KMO and one line with KKMO constructs. The eyes of adults in these lines were brown, and the eggs laid by the transgenic females were also brown. Reverse transcription-polymerase chain reaction（RT-PCR） analysis showed that the A3KMO silkworm lines expressed the transcript in the mid-gut, fat bodies, and Malpighian tubules. The KKMO line expressed the transcript only in the fat bodies and Malpighian tubules. The intensity of eye and egg color in the transgenic lines was proportional to the KMO expression level. Interestingly, transgenic larvae with the A3KMO construct had a light brown larval cuticle, but the KKMO line did not. These results indicate that the wild-type KMO gene can be used as a marker gene for visually screening transgenic silkworms.