Dbh+ catecholaminergic cardiomyocytes contribute to the structure and function of the cardiac conduction system in murine heart.

The heterogeneity of functional cardiomyocytes arises during heart development, which is essential to the complex and highly coordinated cardiac physiological function. Yet the biological and physiological identities and the origin of the specialized cardiomyocyte populations have not been fully comprehended. Here we report a previously unrecognised population of cardiomyocytes expressing Dbhgene encoding dopamine beta-hydroxylase in murine heart. We determined how these myocytes are distributed across the heart by utilising advanced single-cell and spatial transcriptomic analyses, genetic fate mapping and molecular imaging with computational reconstruction. We demonstrated that they form the key functional components of the cardiac conduction system by using optogenetic electrophysiology and conditional cardiomyocyte Dbh gene deletion models. We revealed their close relationship with sympathetic innervation during cardiac conduction system formation. Our study thus provides new insights into the development and heterogeneity of the mammalian cardiac conduction system by revealing a new cardiomyocyte population with potential catecholaminergic endocrine function.

Single-cell RNA sequencing of murine hearts for studying the development of the cardiac conduction system.

The development of the cardiac conduction system (CCS) is essential for correct heart function. However, critical details on the cell types populating the CCS in the mammalian heart during the development remain to be resolved. Using single-cell RNA sequencing, we generated a large dataset of transcriptomes of ~0.5 million individual cells isolated from murine hearts at six successive developmental corresponding to the early, middle and late stages of heart development. The dataset provides a powerful library for studying the development of the heart's CCS and other cardiac components. Our initial analysis identified distinct cell types between 20 to 26 cell types across different stages, of which ten are involved in forming the CCS. Our dataset allows researchers to reuse the datasets for data mining and a wide range of analyses. Collectively, our data add valuable transcriptomic resources for further study of cardiac development, such as gene expression, transcriptional regulation and functional gene activity in developing hearts, particularly the CCS.

Natural variation of Dt2 determines branching in soybean.

Shoot branching is fundamentally important in determining soybean yield. Here, through genome-wide association study, we identify one predominant association locus on chromosome 18 that confers soybean branch number in the natural population. Further analyses determine that Dt2 is the corresponding gene and the natural variations in Dt2 result in significant differential transcriptional levels between the two major haplotypes. Functional characterization reveals that Dt2 interacts with GmAgl22 and GmSoc1a to physically bind to the promoters of GmAp1a and GmAp1d and to activate their transcription. Population genetic investigation show that the genetic differentiation of Dt2 display significant geographic structure. Our study provides a predominant gene for soybean branch number and may facilitate the breeding of high-yield soybean varieties.

A functionally divergent SOC1 homolog improves soybean yield and latitudinal adaptation.

Soybean (Glycine max) grows in a wide range of latitudes, but it is extremely sensitive to photoperiod, which reduces its yield and ability to adapt to different environments. Therefore, understanding of the genetic basis of soybean adaptation is of great significance for breeding and improvement. Here, we characterized Tof18 (SOC1a) that conditions early flowering and growth habit under both short-day and long-day conditions. Molecular analysis confirmed that the two SOC1 homologs present in soybeans (SOC1a and SOC1b) underwent evolutionary functional divergence, with SOC1a having stronger effects on flowering time and stem node number than SOC1b due to transcriptional differences. soc1a soc1b double mutants showed stronger functional effects than either of the single mutants, perhaps due to the formation of SOC1a and SOC1b homodimers or heterodimers. Additionally, Tof18/SOC1a improves the latitudinal adaptation of cultivated soybeans, highlighting the functional importance of SOC1a. The Tof18G allele facilitates adaptation to high latitudes, whereas Tof18A facilitates adaptation to low latitudes. We demonstrated that SOC1s contribute to floral induction in both leaves and shoot apex through inter-regulation with FTs. The SOC1a-SOC1b-Dt2 complex plays essential roles in stem growth habit by directly binding to the regulatory sequence of Dt1, making the genes encoding these proteins potential targets for genome editing to improve soybean yield via molecular breeding. Since the natural Tof18A allele increases node number, introgressing this allele into modern cultivars could improve yields, which would help optimize land use for food production in the face of population growth and global warming.

Parallel selection of distinct Tof5 alleles drove the adaptation of cultivated and wild soybean to high latitudes.

Photoperiod responsiveness is a key factor limiting the geographic distribution of cultivated soybean and its wild ancestor. In particular, the genetic basis of the adaptation in wild soybean remains poorly understood. In this study, by combining whole-genome resequencing and genome-wide association studies we identified a novel locus, Time of Flowering 5 (Tof5), which promotes flowering and enhances adaptation to high latitudes in both wild and cultivated soybean. By genomic, genetic and transgenic analyses we showed that Tof5 encodes a homolog of Arabidopsis thaliana FRUITFULL (FUL). Importantly, further analyses suggested that different alleles of Tof5 have undergone parallel selection. The Tof5H1 allele was strongly selected by humans after the early domestication of cultivated soybean, while Tof5H2 allele was naturally selected in wild soybean, and in each case facilitating adaptation to high latitudes. Moreover, we found that the key flowering repressor E1 suppresses the transcription of Tof5 by binding to its promoter. In turn, Tof5 physically associates with the promoters of two important FLOWERING LOCUS T (FT), FT2a and FT5a, to upregulate their transcription and promote flowering under long photoperiods. Collectively, our findings provide insights into how wild soybean adapted to high latitudes through natural selection and indicate that cultivated soybean underwent changes in the same gene but evolved a distinct allele that was artificially selected after domestication.

A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts.

Pathological hypertrophy underlies sudden cardiac death due to its high incidence of occurrence of ventricular arrhythmias. The alteration of transmural electrophysiological properties in hypertrophic cardiac murine tissue has never been explored previously. In this dataset, we have for the first time conducted high-throughput simultaneous optical imaging of transmembrane potential and calcium transients (CaT) throughout the entire hypertrophic murine hearts at high temporal and spatial resolution. Using ElectroMap, we have conducted multiple parameters analysis including action potential duration/calcium transient duration, conduction velocity, alternans and diastolic interval. Voltage-calcium latency was measured as time difference between action potential and CaT peak. The dataset therefore provides the first high spatial resolution transmural electrophysiological profiling of the murine heart, allowing interrogation of mechanisms driving ventricular arrhythmias associated with pathological hypertrophy. The dataset allows for further reuse and detailed analyses of geometrical, topological and functional analyses and reconstruction of 2-dimensional and 3-dimentional models.

Genetic basis and adaptation trajectory of soybean from its temperate origin to tropics.

Soybean (Glycine max) serves as a major source of protein and edible oils worldwide. The genetic and genomic bases of the adaptation of soybean to tropical regions remain largely unclear. Here, we identify the novel locus Time of Flowering 16 (Tof16), which confers delay flowering and improve yield at low latitudes and determines that it harbors the soybean homolog of LATE ELONGATED HYPOCOTYL (LHY). Tof16 and the previously identified J locus genetically additively but independently control yield under short-day conditions. More than 80% accessions in low latitude harbor the mutations of tof16 and j, which suggests that loss of functions of Tof16 and J are the major genetic basis of soybean adaptation into tropics. We suggest that maturity and yield traits can be quantitatively improved by modulating the genetic complexity of various alleles of the LHY homologs, J and E1. Our findings uncover the adaptation trajectory of soybean from its temperate origin to the tropics.

A flowering time locus dependent on E2 in soybean.

Soybean [Glycine max (L.) Merrill] is very sensitive to changes in photoperiod as a typical short-day plant. Photoperiodic flowering influences soybean latitudinal adaptability and yield to a considerable degree. Identifying new quantitative trait loci (QTLs) controlling flowering time is a powerful initial approach for elucidating the mechanisms underlying flowering time and adaptation to different latitudes in soybean. In this study, we developed a Recombinant Inbred Lines (RILs) population and recorded flowering time under natural long-day conditions. We also constructed a high-density genetic map by genotyping-by-sequencing and used it for QTL mapping. In total, we detected twelve QTLs, four of which are stable and named by qR1-2, qR1-4, qR1-6.1, and qR1-10, respectively. Among these four QTLs, qR1-4 and qR1-6.1 are novel. QTL mapping in two sub-populations classified by the genotype of the maturity locus E2, genetic interaction evaluation between E2 and qR1-2, and qRT-PCR indicated that E2 has an epistatic effect on qR1-2, and that causal gene of qR1-2 acts upstream of E2. We presumed the most likely candidate genes according to the resequencing data and briefly analyzed the geographic distributions of these genes. These findings will be beneficial for our understanding of the mechanisms underlying photoperiodic flowering in soybean, contribute to further investigate of E2, and provide genetic resources for molecular breeding of soybean. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01224-1.

Rapid excavating a FLOWERING LOCUS T-regulator NF-YA using genotyping-by-sequencing.

Soybean (Glycine max (L.) Merrill) is one of the most important crop plants in the world as an important source of protein for both human consumption and livestock fodder. As flowering time contributes to yield, finding new QTLs and further identifying candidate genes associated with various flowering time are fundamental to enhancing soybean yield. In this study, a set of 120 recombinant inbred lines (RILs) which was developed from a cross of two soybean cultivars, Suinong4 (SN4) and ZK168, were genotyped by genotyping-by-sequencing (GBS) approach and phenotyped to expand the cognitive of flowering time by quantitative trait loci (QTL) analysis. Eventually, three stable QTLs related to flowering time which were detected separately located on chromosome 14, 18, and 19 under long-day (LD) conditions. We predicted candidate genes for each QTL and carried out association analyses between the putative causal alleles and flowering time. Moreover, a transient transfection assay was performed and showed that NUCLEAR FACTOR YA 1b (GmNF-YA1b) as a strong candidate for the QTL on chromosome 19 might affect flowering time by suppressing the expression of FLOWERING LOCUS T (GmFT) genes in soybean. QTLs detected in this study would provide fundamental resources for finding candidate genes and clarify the mechanisms of flowering which would be helpful for breeding novel high-yielding soybean cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01237-w.

Natural variation of the Dt2 promoter controls plant height and node number in semi-determinant soybean.

Soybean (Glycine max (L.) Merrill) is an important legume crop worldwide. Plant height (PH) is a quantitative trait that is closely related to node number (NN) and internode length (IL) on the main stem, which together affect soybean yield. To identify candidate genes controlling these three traits in soybean, we examined a recombinant inbred line (RIL) population derived from a cross between two soybean varieties with semi-determinate stems (Dt1Dt1Dt2Dt2), JKK378 and HXW. A quantitative trait locus (QTL) named qPH18 was identified that simultaneously controls PH, NN, and IL; this region harbors the semi-determinant gene Dt2. Sequencing of the Dt2 promoter from JKK378 identified three polymorphisms relative to HXW, including two single nucleotide polymorphism (SNPs) and an 18-bp insertion/deletion polymorphism (Indel). Dt2 expression was lower in the qPH18JKK378 group than in the qPH18HXW group, whereas the expression level of the downstream gene Dt1 showed the opposite tendency. A transient transfection assay confirmed that Dt2 promoter activity is lower in JKK378 compared to HXW. We propose that the polymorphisms in the dominant Dt2 promoter underlie the differences in Dt2 expression and its downstream gene Dt1 in the two parents, thereby affecting PH, NN, IL, and grain weight per plant without altering stem growth habit. Compared to the PH18HXW allele, the qPH18JKK378 allele suppresses Dt2 expression, which releases the inhibition of Dt1 expression, thus enhancing NN and grain yield. Our findings shed light on the mechanism underlying NN and PH in soybean and provide a molecular marker to facilitate breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01235-y.

Weight Function Method for Stress Intensity Factors of Semi-Elliptical Surface Cracks on Functionally Graded Plates Subjected to Non-Uniform Stresses.

In this paper, a weight function method based on the first four terms of a Taylor's series expansion is proposed to determine the stress intensity factors of functionally graded plates with semi-elliptical surface cracks. Cracked surfaces that are subjected to constant, linear, parabolic and cubic stress fields are considered. The weight functions for the surface, deepest and general points on the crack faces of long and deep cracked functionally graded plates are derived, which has never been done before in the literature. The accuracy of the method in this study is then validated by comparing the results with those of finite element modeling. The numerical results indicate that the derived weight functions are highly accurate and robust enough to predict the stress intensity factors for cracked functionally graded plates subjected to non-uniform stress distributions. The weight function method is therefore a time-saving technique and suitable for handling non-uniform stress fields.

CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean.

BACKGROUND: Soybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants. RESULTS: Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL (LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies. CONCLUSIONS: Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35 days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.

A Protocol for Dual Calcium-Voltage Optical Mapping in Murine Sinoatrial Preparation With Optogenetic Pacing.

Among the animal models for studying the molecular basis of atrial and sinoatrial node (SAN) biology and disease, the mouse is a widely used species due to its feasibility for genetic modifications in genes encoding ion channels or calcium handling and signaling proteins in the heart. It is therefore highly valuable to develop robust methodologies for studying SAN and atrial electrophysiological function in this species. Here, we describe a protocol for performing dual calcium-voltage optical mapping on mouse sinoatrial preparation (SAP), in combination with an optogenetic approach, for studying SAP membrane potential, intracellular Ca2+ transients, and pacemaker activity. The protocol includes the details for preparing the intact SAP, robust tissue dual-dye loading, light-programmed pacing, and high-resolution optical mapping. Our protocol provides an example of use of the combination of optogenetic and optical mapping techniques for investigating SAP membrane potential and intracellular Ca2+ transients and pacemaker activity with high temporal and spatial resolution in specific cardiac tissues. Thus, our protocol provides a useful tool for studying SAP physiology and pathophysiology in mice.