Single-Cell Transcriptome Analysis Reveals Dynamic Populations of Vascular Cells in Neointimal Hyperplasia.

BACKGROUND: Neointimal hyperplasia (NIH) is the pathological basis of vascular injury disease. Vascular cells are the dominant cells in the process of NIH, but the extent of heterogeneity amongst them is still unclear. METHODS: A mouse model of NIH was constructed by inducing carotid artery ligation. Single-cell sequencing was then used to analyze the transcriptional profile of vascular cells. Cluster features were determined by functional enrichment analysis, gene set scoring, pseudo-time analysis, and cell-cell communication analysis. Additionally, immunofluorescence staining was conducted on vascular tissues from fibroblast lineage-traced (PdgfraDreER-tdTomato) mice to validate the presence of Pecam1+Pdgfra+tdTomato+ cells. RESULTS: The left carotid arteries (ligation) were compared to right carotid arteries (sham) from ligation-induced NIH C57BL/6 mice. Integrative analyses revealed a high level of heterogeneity amongst vascular cells, including fourteen clusters and seven cell types. We focused on three dominant cell types: endothelial cells (ECs), vascular smooth muscle cells (vSMCs), and fibroblasts. The major findings were: (1) four subpopulations of ECs, including ECs4, mesenchymal-like ECs (ECs1 and ECs2), and fibro-like ECs (ECs3); (2) four subpopulations of fibroblasts, including pro-inflammatory Fibs-1, Sca1+ Fibs-2, collagen-producing Fibs-3, and mesenchymal-like Fibs-4; (3) four subpopulations of vSMCs, including vSMCs-1, vSMCs-2, vSMCs-3, and vSMCs-3-derived vSMCs; (4) ECs3 express genes related to extracellular matrix (ECM) remodeling and cell migration, and fibro-like vSMCs showed strong chemokine secretion and relatively high levels of proteases; (5) fibro-like vSMCs that secrete Vegfa interact with ECs mainly through vascular endothelial growth factor receptor 2 (Vegfr2). CONCLUSIONS: This study presents the dynamic cellular landscape within NIH arteries and reveals potential relationships between several clusters, with a specific focus on ECs3 and fibro-like vSMCs. These two subpopulations may represent potential target cells for the treatment of NIH.

QTL Analysis for Rice Quality-Related Traits and Fine Mapping of qWCR3.

The quality of rice, evaluated using multiple quality-related traits, is the main determinant of its market competitiveness. In this study, two japonica rice varieties with significant differences in quality-related traits were used as parents to construct two populations, BC3F2 and BC3F2:3, with Kongyu131 (KY131) as the recurrent parent. A genetic linkage map was constructed using the BC3F2 population based on 151 pairs of SSR/InDel polymorphic markers selected between the parents. Grain-shape-related traits (grain length GL, grain width GW, and length-to-width ratio LWR), chalkiness-related traits (white-core rate WCR, white-belly rate WBR, white-back rate BR, and chalkiness rate CR), and amylose content (AC) were investigated in the two populations in 2017 and 2018. Except for BR and CR, the traits showed similar characteristics with a normal distribution in both populations. Genetic linkage analysis was conducted for these quality-related traits, and a total of 37 QTLs were detected in the two populations. Further validation was performed on the newly identified QTLs with larger effects, and three grain shape QTLs and four chalkiness QTLs were successfully validated in different environments. One repeatedly validated QTL, qWCR3, was selected for fine mapping and was successfully narrowed down to a 100 kb region in which only two genes, LOC_0s03g45210 and LOC_0s03g45320, exhibited sequence variations between the parents. Furthermore, the variation of LOC_Os03g45210 leads to a frameshift mutation and premature protein termination. The results of this study provide a theoretical basis for positional cloning of the qWCR3 gene, thus offering new genetic resources for rice quality improvement.

QTL detection for rice grain storage protein content and genetic effect verifications.

Rice grain quality is a multifarious attribute mainly governed by multiple nutritional factors. Grain protein is the central component of rice grain nutrition dominantly affecting eating-cooking qualities. Grain protein content is quantitatively influenced by its protein fractions. Genetic quantification of five protein fractions-albumins, globulins, prolamins, glutelin, and grain protein content-were evaluated by exploiting two BC3F2 mapping populations, derived from Kongyu131/TKM9 (population-I) and Kongyu131/Bg94-1 (population-II), which were grown in a single environment. Correlation studies among protein fractions and grain protein content were thoroughly investigated. A genetic linkage map was developed by using 146 single sequence repeat (SSR) markers in population-I and 167 markers in population-II. In total, 40 QTLs were delineated for five traits in both populations. Approximately 22 QTLs were dissected in population-I, derived from Kongyu131/TKM9, seven QTLs for albumin content, four QTLs for globulin content, three QTLs for prolamin content, four QTLs for glutelin content, and four QTLs for grain protein content. In total, 18 QTLs were detected in population-II, derived from Kongyu131/Bg94-1, five QTLs for albumin content, three QTLs for globulin content, four QTLs for prolamin content, two QTLs for glutelin content, and four QTLs for grain protein content. Three QTLs, qAlb7.1, Alb7.2, and qGPC7.2, derived from population-II (Kongyu131/Bg94-1) for albumin and grain protein content were successfully validated in the near isogenic line (NIL) populations. The localized chromosomal locus of the validated QTLs could be helpful for fine mapping via map-based cloning to discover underlying candidate genes. The functional insights of the underlying candidate gene would furnish novel perceptivity for the foundation of rice grain protein content and trigger the development of nutritionally important rice cultivars by combining marker-assisted selection (MAS) breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01436-7.

Mitochondrial activity regulates the differentiation of skin-derived mesenchymal stem cells into brown adipocytes to contribute to hypertension.

BACKGROUND: Brown adipocytes (BAs) are major components of brown adipose tissue (BAT), which is involved in blood pressure regulation. BAs are derived from multiple progenitors, including PDGFRα+ adipose-derived stem cells (ASCs). Skin-derived mesenchymal stem cells (S-MSCs) have the capacity to differentiate into adipocytes; however, their ability to differentiate into BAs remains unexplored. We aim to study the ability and regulatory mechanism of the differentiation of S-MSCs into BAs and the direct role of BAT in blood pressure regulation. METHODS: Protein expression was measured by flow cytometry or Western blotting, and gene mRNA levels were quantified by real-time quantitative PCR (RT-PCR). To induce the differentiation of S-MSCs into BAs, S-MSCs were stimulated with a brown adipogenic cocktail comprising insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was determined by flow cytometry and fluorescence staining. Hypertension was induced in WT mice by infusion of angiotensin II (Ang II), and systolic blood pressure (SBP) was measured using a tail cuff. Interscapular brown adipose tissue (iBAT)-deficient mice were generated by surgical removal of the iBAT depot, after which the animals were allowed to recover for 6 days. Aortic, iBAT, and heart tissue sections were analyzed by hematoxylin and eosin (HE) staining. RESULTS: We found that in vitro, S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during differentiation of S-MSCs into BAs. In vivo, iBAT was converted to white adipose tissue (WAT) in Ang II-induced hypertensive mice. We assessed the direct role of BAT in blood pressure (BP) regulation by using iBAT-deficient mice (generated by surgical removal of iBAT) and C57BL/6 (wild-type (WT)) mice and found that Ang II-induced BP elevation and vascular damage were markedly aggravated in iBAT-deficient mice compared with WT mice. CONCLUSIONS: This study demonstrates that PDGFRα+ S-MSCs are able to differentiate into BAs and that this differentiation is regulated by mitochondrial activity. We also show that BAT plays a direct role in ameliorating Ang II-induced hypertension. The therapeutic potential of BAT for the prevention of hypertension-induced organ remodeling thus warrants further investigation.

Gene diagnosis and targeted breeding for blast-resistant Kongyu 131 without changing regional adaptability.

The fungus Magnaporthe oryzae threatens the rice production of Kongyu 131 (KY131), a leading japonica variety in Northeast China. In this study, two rice lines, KP1 and KP2-Hd1, were obtained by introgressing the blast resistance genes Pi1 and Pi2 into KY131, respectively. However, both lines headed later than KY131. RICE60K SNP array analysis showed that Hd1 closely linked to Pi2 was introgressed into KP2-Hd1, and the linkage drag of Hd1 was broken by recombination. On the other hand, no known flowering genes were introgressed into KP1. Gene diagnosis by resequencing six flowering genes showed that KP1 carried functional Hd16 and Ghd8 alleles. Due to its suppression role in heading under long-day conditions, Ghd8 was chosen as the target for gene editing to disrupt its function. Four sgRNAs targeting different sites within Ghd8 were utilized to induce large-deletion mutations, which were easy to detect via agarose gel electrophoresis. All the ghd8-mutated KP1 lines were resistant to rice blast disease and headed earlier than the control KP1, even than KY131, under natural long-day conditions, which ensures its growth in Northeast China. This study confirmed that a combination of gene diagnosis and targeted gene editing is a highly efficient way to quickly eliminate undesired traits in a breeding line.

Dissecting combining ability effect in a rice NCII-III population provides insights into heterosis in indica-japonica cross.

BACKGROUND: Combining ability is a measure for selecting elite parents that make the highest contributions to hybrid performance. However, the genetic bases of combining ability and how they contributed to heterosis is seldomly known. RESULTS: We constructed a both NCII and NCIII population derived from an indica-japonica cross to study the relationship among parental performance, combining ability and hybrid performance of 11 agronomic traits. Among them, specific combining ability is more important to grain yield than parental performance and general combining ability. We performed linkage analyses to phenotypic values and combining ability of all 11 traits in Doubled haploid lines and its two backcross populations and identified 108 QTLs in total. Among these QTLs, four known loci, Sd1, Ghd7, Ghd8 and DEP1 contribute a lot to GCA effects of agronomic traits except grain yield and seed setting rate. Three QTLs, Ghd8, S5 and qS12, contribute a lot to SCA effects of grain yield and present overdominace. CONCLUSIONS: Our study provides insights into the genetic bases of combining ability and heterosis and will promote the improvements of indica-japonica hybrid breeding.

QTL analysis on rice grain appearance quality, as exemplifying the typical events of transgenic or backcrossing breeding.

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F9-10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.

Comparative mapping of chalkiness components in rice using five populations across two environments.

BACKGROUND: Chalkiness is a major constraint in rice production because it is one of the key factors determining grain quality (appearance, processing, milling, storing, eating, and cooking quality) and price. Its reduction is a major goal, and the primary purpose of this study was to dissect the genetic basis of grain chalkiness. Using five populations across two environments, we also sought to determine how many quantitative trait loci (QTL) can be consistently detected. We obtained an integrated genetic map using the data from five mapping populations and further confirmed the reliability of the identified QTL. RESULTS: A total of 79 QTL associated with six chalkiness traits (chalkiness rate, white core rate, white belly rate, chalkiness area, white core area, and white belly area) were mapped on 12 chromosomes using five populations (two doubled haploid lines and three recombinant inbred lines) across two environments (Hainan in 2004 and Wuhan in 2004). The final integrated map included 430 markers; 58.3% of the QTL clustered together (QTL clusters), 71.4% of the QTL clusters were identified in two or more populations, and 36.1% of the QTL were consistently detected in the two environments. The QTL could be detected again and showed dominance (qWBR1, qWBR8, qWBR12, and qCR5) or overdominance effects (qWCR7) for the rate of the white belly or white core, respectively, and all four QTL clusters derived from Zhenshan 97 controlling white belly rate were stably and reliably identified in an F2 population. CONCLUSIONS: Our results identified 79 QTL associated with six chalkiness traits using five populations across two environments and yielded an integrated genetic map, indicating most of the QTL clustered together and could be detected in different backgrounds. The identified QTL were stable and reliable in the F2 population, and they may facilitate our understanding of the QTL related to chalkiness traits in different populations and various environments, the relationships among the various chalkiness QTL, and the genetic basis for chalkiness. Thus, our results may be immediately used for map-based cloning of important QTL and in marker-assisted breeding to improve grain quality in rice breeding.

Analysis of quantitative trait loci affecting chlorophyll content of rice leaves in a double haploid population and two backcross populations.

Chlorophyll content, one of the most important physiological parameters related to plant photosynthesis, is usually used to predict yield potential. To map the quantitative trait loci (QTLs) underlying the chlorophyll content of rice leaves, a double haploid (DH) population was developed from an indica/japonica (Zhenshan 97/Wuyujing 2) crossing and two backcross populations were established subsequently by backcrossing DH lines with each of their parents. The contents of chlorophyll a and chlorophyll b were determined by using a spectrophotometer to directly measure the leaf chlorophyll extracts. To determine the leaf chlorophyll retention along with maturation, all measurements were performed on the day of heading and were repeated 30 days later. A total of 60 QTLs were resolved for all the traits using these three populations. These QTLs were distributed on 10 rice chromosomes, except chromosomes 5 and 10; the closer the traits, the more clustering of the QTLs residing on common rice chromosomal regions. In general, the majority of QTLs that specify chlorophyll a content also play a role in determining chlorophyll b content. Strangely, chlorophyll content in this study was found mostly to be lacking or to have a negative correlation with yield. In both backcross F1 populations, overdominant (or underdominant) loci were more important than complete or partially dominant loci for main-effect QTLs and epistatic QTLs, thereby supporting previous findings that overdominant effects are the primary genetic basis for depression in inbreeding and heterosis in rice.

Use of serial analysis of gene expression to reveal the specific regulation of gene expression profile in asthmatic rats treated by acupuncture.

BACKGROUND: Asthma has become an important public health issue and approximately 300 million people have suffered from the disease worldwide. Nowadays, the use of acupuncture in asthma is increasing. This study intended to systematically analyze and compare the gene expression profiles between the asthmatic and acupuncture-treated asthmatic rat lung, and tried to gain insight into the molecular mechanism underlying the early airway response (EAR) phase of asthma treated by acupuncture. METHODS: Four tag libraries of serial analysis of gene expression (SAGE) were established from lung tissues of control rats (CK), asthmatic rats (AS), asthmatic rats treated by acupuncture (ASAC), and control rats treated by acupuncture (CKAC). Bioinformatic analyses were carried out by using the methods including unsupervised hierarchical clustering, functional annotation tool of the database for annotation, visualization, and integrated discovery (DAVID), gene ontology (GO) tree machine, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. RESULTS: There were totally 186 differentially expressed tags (P < 0.05, P(CK/AS)) between the libraries of CK and AS, 130 differentially expressed tags between libraries of AS/ASAC (P < 0.05, P(AS/ASAC)), and 144 differentially expressed tags between libraries of CK/CKAC (P < 0.05, P(CK/CKAC)). The gene expression profiles of AS and ASAC were more similar than other libraries via unsupervised SAGE clustering. By comparison of P(CK/AS) and P(AS/ASAC), the DAVID genes functional classification was found to be changed from "immune response" to "response to steroid hormone stimulus", and the GO term "antigen processing and presentation of peptide antigen" disappeared in P(AS/ASAC). Totally 3 same KEGG pathways were found among the three groups. Moreover, 21 specific tags of the acupuncture in treating asthma were detected using Venn diagrams. CONCLUSION: Our SAGE research indicates that the gene expression profile of the EAR phase of asthma could be effectively and specifically regulated by acupuncture, which suggests that the gene expression of immune response and steroid hormone may play an important role in the treatment.

Serial analysis of gene expression in a rat lung model of asthma.

BACKGROUND AND OBJECTIVE: The pathogenesis and molecular mechanism underlying asthma remain undetermined. The purpose of this study was to identify genes and pathways involved in the early airway response (EAR) phase of asthma by using serial analysis of gene expression (SAGE). METHODS: Two SAGE tag libraries of lung tissues derived from a rat model of asthma and controls were generated. Bioinformatic analyses were carried out using the Database for Annotation, Visualization and IntegratedDiscovery Functional Annotation Tool, Gene Ontology (GO) TreeMachine and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. RESULTS: A total of 26 552 SAGE tags of asthmatic rat lung were obtained, of which 12 221 were unique tags. Of the unique tags, 55.5% were matched with known genes. By comparison of the two libraries, 186 differentially expressed tags (P < 0.05) were identified, of which 103 were upregulated and 83 were downregulated. Using the bioinformatic tools these genes were classified into 23 functional groups, 15 KEGG pathways and 37 enriched GO categories. CONCLUSIONS: The bioinformatic analyses of gene distribution, enriched categories and the involvement of specific pathways in the SAGE libraries have provided information on regulatory networks of the EAR phase of asthma. Analyses of the regulated genes of interest may inform new hypotheses, increase our understanding of the disease and provide a foundation for future research.

Characterization of the genetic basis for yield and its component traits of rice revealed by doubled haploid population.

Main-effect QTL, epistatic effects and their interactions with environment are important genetic components of quantitative traits. In this study, we analyzed the QTLs, epistatic effects and QTL by environment interactions (QE) underlying nine traits of yield and yield-component, using a doubled-haploid (DH) population consisted of 190 lines from the cross between an indica parent Zhenshan 97 and a japonica parent WYJ 2, and tested in two-year replicated field trials. A genetic linkage map with 179 SSR (simple sequence repeat) marker loci was constructed. A mixed linear model approach was applied to detect QTLs, digenic interactions and QEs for the nine traits. In total, 57 QTLs of main effects, 41 digenic interactions, eight QEs and seven interactions of epistasis by environment were detected. Each of the main-effect QTLs individually explained 1.3 % to 25.8% of the phenotypic variations. And they collectively explained 11.5% to 66.8% of the phenotypic variations for these traits. Most of the traits (except seed setting) had the QTLs simultaneously detected in two years. Many of the traits shared same QTLs with each other, which is consistent with their significant phenotypic correlations. The pleiotropism or tight linkage of QTLs for different traits might be the important genetic base for trait correlations. The environmental influences on the stability of the trait performance were also discussed.