Introduction Cardiac outflow tract (OFT) development relies on the interactions among cardiac neural crest cells (CNCC) and the second heart field (SHF) cells. However, the molecular signals underlying these interactions remain largely unclear. Objectives Vinculin (VCL) is an adaptor protein for focal adhesions, which integrates various external signals and mediates cell–cell communications, representing a potential hub gene directing the OFT development. Here, we aimed to build a holistic map of signal flows underlying CNCC-directed OFT development using neural crest-specific Vcl knockout mouse mutants (Vcl cKO). Methods We validated the OFT defects of Vcl cKO mutants by performing various immunohistochemistry analyses. Subsequent high-resolution transcriptomic analysis reconstructed the differentiation trajectory of CNCCs along the vascular smooth muscle (VSMC) lineage. The spatial RNA-seq further aligned the transcriptomes of OFT cells with their spatial patterns to explore the cell interaction dynamics among OFT cells. Exploiting human pluripotent stem cell (hPSC)-based in vitro differentiation assays, the signaling pathways underlying Vcl-mediated CNCC progression along the VSMC lineage were defined. Results Vcl cKO exhibited malformation of pharyngeal arch arteries, OFT septation defects and hyperplastic semilunar valves, due to defects in migration and vascular smooth muscle (VSMC) lineage differentiation of CNCCs, which prohibited the subsequent myocardialization, perturbing the OFT and valve remodeling. High-resolution transcriptomic analysis revealed that CNCC-to-VSMC differentiation is orchestrated by Vcl-dependent p38 and Tgf-β signalings. Multimodal analysis integrating the single-cell and spatial transcriptomes of OFT cells further suggested a shift of signal flow among OFT cells and loss of cell-extracellular matrix interaction perturb OFT morphogenesis. Conclusion In sum, dynamic signal flows among CNCC and SHF cells guide OFT remodeling, in which p38-Tgf-β signaling mediates the VSMC lineage differentiation of CNCCs, and timely regulates their interactions with SHF-derived cells through ECM.

Chalkiness is an important grain quality trait in rice. Chalk5, encoding a vacuolar H+-translocating pyrophosphatase, is a major gene affecting both the percentage of grains with chalkiness (PGWC) and chalkiness degree (DEC) in rice. Reducing its expression can decrease both PGEC and DEC. In this study, the first exon of Chalk5 was edited in the elite restorer line 9311 using the CRISPR/Cas9 system and two knockout mutants were obtained, one of which did not contain the exogenous Cas9 cassette. PGWC and DEC were both significantly reduced in both mutants, while the seed setting ratio (SSR) was also significantly decreased. Staggered sowing experiments showed that the chalkiness of the mutants was insensitive to temperature during the grain-filling stage, and the head milled rice rate (HMRR) could be improved even under high-temperature conditions. Finally, in the hybrid background, the mutants showed significantly reduced PGWC and DEC without changes in other agronomic traits. The results provide important germplasm and allele resources for breeding high-yield rice varieties with superior quality, especially for high-yield indica hybrid rice varieties with superior quality in high-temperature conditions.

The morphological characteristics of the rice panicle play a pivotal role in influencing yield. In our research, we employed F2 and F2:3 populations derived from the high-yielding hybrid rice variety Chaoyou 1000. We screened 123 pairs of molecular markers, which were available, to construct the genetic linkage map. Subsequently, we assessed the panicle morphology traits of F2 populations in Lingshui County, Hainan Province, in 2017, and F2:3 populations in Hangzhou City, Zhejiang Province, in 2018. These two locations represent two types of ecology. Hangzhou's climate is characterized by high temperatures and humidity, while Lingshui's climate is characterized by a tropical monsoon climate. In total, 33 QTLs were identified, with eight of these being newly discovered, and two of them were consistently detected in two distinct environments. We identified fourteen QTL-by-environment interactions (QEs), which collectively explained 4.93% to 59.95% of the phenotypic variation. While most of the detected QTLs are consistent with the results of previous tests, the novel-detected QTLs will lay the foundation for rice yield increase and molecular breeding.

As a temperature-loving crop, rice is sensitive to low temperatures. With the popularization of direct-seeded rice, cold tolerance (CT) at the bud burst stage has become an important breeding goal. Here, we evaluated CT for 513 rice accessions at the bud burst stage. A total of 13 QTLs were detected by genome-wide association analysis using the severity of damage (SD) and survival rate of seedlings (SR) as indicators of CT. Based on analyses of LD blocks, GO enrichment, gene expression and haplotype, we identified five genes, LOC_Os01g35184, LOC_Os01g56150, LOC_Os01g73410, LOC_Os02g36740, and LOC_Os09g28180, as the most likely candidates for qSD1-1, qSD1-4, qSD1-5, qSD2-1, and qSR9-1, respectively, for CT. The accumulative effects of favorable haplotypes for the above five most likely candidate genes played an important role in the improvement of the CT of rice cultivars. Hence, this study has furnished valuable insights for advancing gene cloning and pyramiding breeding, aiming to enhance cold tolerance during the bud burst stage in rice.

再生稻具有较好的外观品质.为解析再生稻外观品质的遗传基础,本研究利用籼稻明恢63和粳稻02428构建的双向导入系为材料,连续两年在湖北荆州考察了双向导入系头季和再生季的外观品质.结果表明,明恢63头季和再生季的外观品质均显著优于02428,双向导入系的所有性状均表现为连续分布.同一性状在头季和再生季间表现为显著正相关,同一季节内不同性状间也表现出显著的相关性,其中粒宽对外观品质的影响最大.结合双向导入系已有的4568个Bin的高密度基因型数据,共定位到57个影响再生稻外观品质的数量性状点位(QTL),位于全部12条染色体上.其中25个QTL在两年间稳定表达,17个QTL在两个遗传背景下被重复鉴定到.此外,第3号染色体16.28～17.33 Mb、第5号染色体3.35～4.28 Mb、第7号染色体24.68～25.46 Mb和第11号染色体6.19～6.97 Mb这4个区间同时影响4个以上性状,来自明恢63的等位基因在这4个区间均可提高外观品质.最后,利用分离群体验证了第11号染色体6.19～6.97 Mb具有真实性.本研究结果为分子育种改良再生稻的外观品质提供了遗传基础.

single-cell RNA-seq data analysis: all.cncc.combined.EMBO.mapped.Rdata: public CNCC single-cell RNA-seq data integration E13.5_CNCC_merged_updated.Rdata: single-cell RNA-seq data of E13.5 CNCC generated in Elly lab all.seurat.GFP.Rdata: scRNA-seq data with GFP detected visium.merge_AB.control.Rdata: R processed ST data for slice A and B visium.merge_CD.mutant.Rdata: R processed ST data for slice A and B

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges. Single-cell profiling studies of the human gastrointestinal tract are increasing, offering an excellent opportunity to generate the first Human Gut Cell Atlas. This Roadmap presents a structured direction towards this goal and provides a detailed overview of the major challenges.

水稻分蘗角度是水稻株型建成的重要性状之一,对水稻产量有着重要的贡献.目前水稻中可利用的分蘗角度调控基因主要有TAC1(Tiller Angle Control 1)和TIG1(Tiller Inclided Growth 1),需进一步挖掘新的可用基因资源和分子标记以促进水稻理想株型育种.本研究中以大角度的野生稻为供体,小角度的栽培稻珍汕97为受体构建了BC3F2群体,在第54号家系中分蘗角度存在分离,利用QTL-seq技术进行水稻分蘗角度的QTL定位,在8号染色体上检测到一个QTL位点.通过对区间内已知基因的序列比对提出TIG1为候选基因.根据TIG1启动子-449 bp处C→T的关键变异设计KASP功能性分子标记,并在定位群体和育成品种中进行了验证,证实利用该KASP标记可以准确地鉴定出TIG1位点的基因型.TIG1在粳稻中以大角度基因型TIG1占绝对优势,而在籼稻中61.40％的品种为小角度基因型tig1,38.40％的品种为大角度基因型TIG1,对水稻株型改良有着重要的潜在利用价值.该KASP标记的开发为水稻分蘗角度的分子标记辅助改良提供了新工具,有望加快水稻理想株型的育种进程.

春两优长70是长江大学、中国农业科学院作物科学研究所与中国农业科学院深圳农业基因组研究所选用籼型两系不育系春6S与恢复系长恢70组配育成的籼型杂交中稻新组合,具有产量高、生育期适中、抗倒伏、米质优等特点.2021年通过国家农作物品种审定委员会审定.

将水稻(鄂香1号)与甜高粱[Sorghum dochna(Forssk.)Snowden]远缘杂交后,经过13代连续自交筛选,获得一批性状相对稳定的变异株系.对其中11个(编号为DC1～DC11)有代表性的变异株系进行表型、遗传背景和应用研究.结果表明,各变异株系生育期(122～141 d)、株高(118.9～228.9 cm)、单株产量(27.5～40.4 g)、结实率(49.7％～89.6％)、垩白度(0.3％～14.4％)、整精米率(53.9％～68.6％)等性状差异明显.利用水稻差异性鉴定的48对标准SSR引物对受体亲本及变异株系进行鉴定,发现该11个株系与鄂香1号差异引物个数为8～15个,表明这11个株系是不同于受体亲本的新材料,该11个株系之间的差异引物个数为2～15个,表明多数变异株系之间也存在差异.利用受体亲本和变异株系与不育系C6S配制杂交组合并进行综合评价,发现部分变异株系所配杂交组合与对照品种丰两优四号相比,在产量和米质方面具有一定的优势.表明通过远缘杂交创造的部分变异株系具有潜在的应用价值.

为解析再生稻蒸煮食味品质的遗传基础,本研究连续两年考察了以籼稻(Indica)'明恢63'和粳稻(Japonica)'02428'构建的双向导入系的头季和再生季的蒸煮食味品质.结果表明,'明恢63'的头季直链淀粉含量(amylose content in the first season,FAC)和头季碱消值(alkali spread value in the first season,FASV)与'02428'有显著差异,其再生季胶稠度(gel consistency in the ratoon season,RGC)与再生季碱消值(alkali spread value in the ratoon season,RASV)与'02428'有显著差异,最终'明恢63'的直链淀粉含量变化率(amylose content ratio,ACR)和碱消值变化率(alkali spread value ratio,ASVR)与'02428'有显著差异.双向导入系的所有性状均表现为连续分布,同一性状在两季间表现出显著正相关.结合双向导入系高密度基因型数据,共定位到30个影响再生稻蒸煮食味品质的数量性状位点(quantitative trait locus,QTL),其中7个QTL在两年间稳定表达,1个QTL在头季和再生季被重复鉴定到,5个QTL在两个遗传背景下稳定表达.最后,利用分离群体验证了第11号染色体6.19～6.97 Mb的真实性.本研究结果为分子育种改良再生稻的蒸煮食味品质提供了基因资源.

Hirschsprung disease is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells, leading to intestinal obstruction. Here, using induced pluripotent stem cell-based models of Hirschsprung and single-cell transcriptomic analysis, we identify a gene set of 118 genes commonly dysregulated in all patient enteric neural crest cells, and suggest HDAC1 may be a key regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events are associated with severe form of Hirschsprung. In particular, the higher inclusion rate of exon 9 in PTBP1 and the perturbed expression of a PTBP1-target, PKM, are significantly enriched in these patient cells, and associated with the defective oxidative phosphorylation and impaired neurogenesis. Hedgehog-induced oxidative phosphorylation significantly enhances the survival and differentiation capacity of patient cells. In sum, we define various factors associated with Hirschsprung pathogenesis and demonstrate the implications of oxidative phosphorylation in enteric neural crest development and HSCR pathogenesis.

Additional file 2. Anonymized genetic testing results of the 54 patients newly reported in the current study.

Retrotrapezoid nucleus (RTN) neurons in the brainstem regulate the ventilatory response to hypercarbia. It is unclear how PHOX2Bpolyalanine repeat mutations (PHOX2B-PARMs) alter the function of PHOX2B and perturb the formation of RTN neurons. Here, we generated human brainstem organoids (HBSOs) with RTN-like neurons from human pluripotent stem cells. Single-cell transcriptomics revealed that expression of PHOX2B+7Ala PARM alters the differentiation trajectories of the hindbrain neurons and hampers the formation of the RTN-like neurons in HBSOs. With the unguided cerebral organoids (HCOs), PHOX2B+7Ala PARM interrupted the patterning of PHOX2B+ neurons with dysregulation of Hedgehog pathway and HOX genes. With complementary use of HBSOs and HCOs with a patient and two mutant induced pluripotent stem cell lines carrying different polyalanine repetition in PHOX2B, we further defined the association between the length of polyalanine repetition and malformation of RTN-respiratory center and demonstrated the potential toxic gain of function of PHOX2B-PARMs, highlighting the uniqueness of these organoid models for disease modeling.

Abstract Hirschsprung disease (HSCR) is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells (ENCCs), leading to intestinal obstruction. Here, using iPSC-based models of HSCR and single-cell transcriptomic analysis, we identified a core gene set of 118 genes commonly dysregulated in all HSCR-ENCCs, with HDAC1 found to be a master regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events were associated with severe form of HSCR. In particular, the higher inclusion rate of exon 9 in PTBP1 and the perturbed expression of a PTBP1-target, PKM, were significantly enriched in these HSCR-ENCCs, and associated with the defective oxidative-phosphorylation (OXPHOS) and impaired neurogenesis. Hedgehog-induced OXPHOS significantly enhanced the survival and differentiation capacity of HSCR-ENCCs. In sum, we have defined the core factors underpinning HSCR disease and demonstrated the implications of OXPHOS in ENCC development and HSCR pathogenesis.

library(Seurat) # seurat object for vcl ENCC print(load("keyRdata/vcl.encc.integrated.Rdata")) # annotation subtypes in each lineage table(vcl.combined$lineage.sub) # condition, normal vs Vcl cKO ENCC table(vcl.combined$stage) ####################### # seurat object for vcl CNCC print(load("E13.5_CNCC_merged_updated.Rdata")) # sample, ct2 is control and vcl is Vcl cKO CNCC table(seuset$orig.ident) # cell annotation file # unsupervised clusters, see more information in HKU thesis table(all_tsne$ident)

OsVP1 and Sdr4 play an important role in regulating seed dormancy that involved in multiple metabolism and regulatory pathways. Seed dormancy and germination are critical agricultural traits influencing rice grain yield. Although there are some genes have identified previously, the comprehensive understanding based on transcriptome is still deficient. In this study, we generated mutants of two representative regulators of seed germination, Oryza sativa Viviparous1 (OsVP1) and Seed dormancy 4 (Sdr4), by CRISPR/Cas9 approach and named them cr-osvp1 and cr-sdr4. The weakened dormancy of mutants indicated that the functions of OsVP1 and Sdr4 are required for normal early seed dormancy. There were 4157 and 8285 differentially expressed genes (DEGs) were identified in cr-osvp1 vs. NIP and cr-sdr4 vs. NIP groups, respectively, with a large number of overlapped DEGs between two groups. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of common DEGs in two groups showed that genes related to carbohydrate metabolic, nucleoside metabolic, amylase activity and plant hormone signal transduction were involved in the dormancy regulation. These results suggest that OsVP1 and Sdr4 play an important role in regulating seed dormancy by multiple metabolism and regulatory pathways. The systematic analysis of the transcriptional level changes provides theoretical basis for the research of seed dormancy and germination in rice.

Ratooning ability is a key factor that influences ratoon rice yield, in the area where light and temperature are not enough for second season rice. In the present study, an introgression line population derived from Minghui 63 as the recipient parent and 02428 as the donor parent was developed, and a high-density bin map containing 4568 bins was constructed. Nine ratooning-ability-related traits were measured, including maximum tiller number, panicle number, and grain yield per plant in the first season and ratoon season, as well as three secondary traits, maximum tiller number ratio, panicle number ratio, and grain yield ratio. A total of 22 main-effect QTLs were identified and explained for 3.26–18.63% of the phenotypic variations in the introgression line population. Three genomic regions, including 14.12–14.65 Mb on chromosome 5, 4.64–5.76 Mb on chromosome 8, and 10.64–15.52 Mb on chromosome 11, were identified to simultaneously control different ratooning-ability-related traits. Among them, qRA5 in the region of 14.12–14.65 Mb on chromosome 5 was validated for its pleiotropic effects on maximum tiller number and panicle number in the first season, as well as its maximum tiller number ratio, panicle number ratio, and grain yield ratio. Moreover, qRA5 was independent of genetic background and delimited into a 311.16 kb region by a substitution mapping approach. These results will help us better understand the genetic basis of rice ratooning ability and provide a valuable gene resource for breeding high-yield ratoon rice varieties.

With the rapid development of single-cell sequencing technologies, it has become a powerful strategy for the discovery of rare cells and delineating the molecular basis underlying various biological processes. Use of single-cell multimodal sequencing to explore the chromatin accessibility, gene expression and spatial transcriptome has propelled us to success in untangling the unknowns in the enteric nervous system (ENS) and provided unprecedented resources for building new diagnostic framework for enteric neuropathies. Here, we summarize the recent findings of single-cell multimodal sequencing, especially focusing on the most commonly used single-cell RNA sequencing (scRNA-seq) on ENS cells, ranged from the progenitors, neural crest (NC) cells, to the mature ENS circuit, in both human and mouse. These studies have highlighted the heterogeneity of ENS cells at various developmental stages and discovered numerous novel cell types. We will also discuss various computational methods that were used to reconstruct the differentiation trajectories of the developing ENS and to elucidate the cell fate decisions. Profiling disease mechanisms and cellular drug responses with single-cell multimodal omics techniques likely leads to a paradigm shift in the field of biomedical research. Further improvements in the high-resolution sequencing platforms and integrative computational tools will greatly hasten their applications in both the basic and translational medicine.

水稻的茎秆强度影响植株的抗倒伏能力.本研究通过60Coγ射线辐射籼稻'9311',获得了一个茎秆壁增厚的突变体st1,并对突变体进行了表型鉴定和基因定位分析.结果表明,与野生型'9311'相比,突变体st1重心高显著降低,基部第四、五节间缩短退化,株高显著变矮.突变体基部第二节间和基部第三节间厚度分别为1.63和1.75 mm,显著高于野生型,倒伏指数显著降低.茎秆解剖结构表明,st1的基部第二节间的大维管束数目、节间表皮和基本组织厚度显著高于野生型.主要农艺性状分析表明,突变体结实率降低,粒宽显著增加,千粒重、垩白粒率和垩白度显著增大.遗传分析表明,突变体st1的突变性状受1对隐性基因控制.利用Mutmap测序定位st1基因.结果表明,st1基因位于第2染色体.本研究为水稻茎秆壁增厚基因的克隆和功能分析提供科学依据,也为水稻抗倒伏研究提供了良好的种质资源.