Bulk Segregant Analysis Sequencing and RNA-Seq Analyses Reveal Candidate Genes Associated with Sepal Color Phenotype of Eggplant (Solanum melongena L.).

Eggplant is a highly significant vegetable crop and extensively cultivated worldwide. Sepal color is considered one of the major commercial traits of eggplant. Eggplant sepals develop from petals, and sepals have the ability to change color by accumulating anthocyanins, but whether the eggplants in sepal and their biosynthetic pathways are the same as those in petals is not known. To date, little is known about the underlying mechanisms of sepal color formation. In this study, we performed bulked segregant analysis and transcriptome sequencing using eggplant sepals and obtained 1,452,898 SNPs and 182,543 InDel markers, respectively, as well as 123.65 Gb of clean data using transcriptome sequencing. Through marker screening, the genes regulating eggplant sepals were localized to an interval of 2.6 cM on chromosome 10 by bulked segregant analysis sequencing and transcriptome sequencing and co-analysis, combined with screening of molecular markers by capillary electrophoresis. Eight possible candidate genes were then screened to further interpret the regulatory incentives for the eggplant sepal color.

The chromosome-scale reference genome and transcriptome analysis of Solanum torvum provides insights into resistance to root-knot nematodes.

Solanum torvum (Swartz) (2n = 24) is a wild Solanaceae plant with high economic value that is used as a rootstock in grafting for Solanaceae plants to improve the resistance to a soil-borne disease caused by root-knot nematodes (RKNs). However, the lack of a high-quality reference genome of S. torvum hinders research on the genetic basis for disease resistance and application in horticulture. Herein, we present a chromosome-level assembly of genomic sequences for S. torvum combining PacBio long reads (HiFi reads), Illumina short reads and Hi-C scaffolding technology. The assembled genome size is ~1.25 Gb with a contig N50 and scaffold N50 of 38.65 Mb and 103.02 Mb, respectively as well as a BUSCO estimate of 98%. GO enrichment and KEGG pathway analysis of the unique S. torvum genes, including NLR and ABC transporters, revealed that they were involved in disease resistance processes. RNA-seq data also confirmed that 48 NLR genes were highly expressed in roots and fibrous roots and that three homologous NLR genes (Sto0288260.1, Sto0201960.1 and Sto0265490.1) in S. torvum were significantly upregulated after RKN infection. Two ABC transporters, ABCB9 and ABCB11 were identified as the hub genes in response to RKN infection. The chromosome-scale reference genome of the S. torvum will provide insights into RKN resistance.

Induction of Male Sterility by Targeted Mutation of a Restorer-of-Fertility Gene with CRISPR/Cas9-Mediated Genome Editing in Brassica napus L.

Brassica napus L. (canola, oil seed rape) is one of the world's most important oil seed crops. In the last four decades, the discovery of cytoplasmic male-sterility (CMS) systems and the restoration of fertility (Rf) genes in B. napus has improved the crop traits by heterosis. The homologs of Rf genes, known as the restoration of fertility-like (RFL) genes, have also gained importance because of their similarities with Rf genes. Such as a high non-synonymous/synonymous codon replacement ratio (dN/dS), autonomous gene duplications, and a possible engrossment in fertility restoration. B. napus contains 53 RFL genes on chromosomes A9 and C8. Our research aims to study the function of BnaRFL11 in fertility restoration using the CRISPR/Cas9 genome editing technique. A total of 88/108 (81.48%) T0 lines, and for T1, 110/145 (75%) lines carried T-DNA insertions. Stable mutations were detected in the T0 and T1 generations, with an average allelic mutation transmission rate of 81%. We used CRISPR-P software to detect off-target 50 plants sequenced from the T0 generation that showed no off-target mutation, signifying that if the designed sgRNA is specific for the target, the off-target effects are negligible. We also concluded that the mutagenic competence of the designed sgRNAs mediated by U6-26 and U6-29 ranged widely from 31% to 96%. The phenotypic analysis of bnarfl11 revealed defects in the floral structure, leaf size, branch number, and seed production. We discovered a significant difference between the sterile line and fertile line flower development after using a stereomicroscope and scanning electron microscope. The pollen visibility test showed that the pollen grain had utterly degenerated. The cytological observations of homozygous mutant plants showed an anther abortion stage similar to nap-CMS, with a Orf222, Orf139, Ap3, and nad5c gene upregulation. The bnarfl11 shows vegetative defects, including fewer branches and a reduced leaf size, suggesting that PPR-encoding genes are essential for the plants' vegetative and reproductive growth. Our results demonstrated that BnaRFL11 has a possible role in fertility restoration. The current study's findings suggest that CRISPR/Cas9 mutations may divulge the functions of genes in polyploid species and provide agronomically desirable traits through a targeted mutation.

BnA1.CER4 and BnC1.CER4 are redundantly involved in branched primary alcohols in the cuticle wax of Brassica napus.

KEY MESSAGE: The mutations BnA1.CER4 and BnC1.CER4 produce disordered wax crystals types and alter the composition of epidermal wax, causing increased cuticular permeability and sclerotium resistance. The aerial surfaces of land plants are coated with a cuticle, comprised of cutin and wax, which is a hydrophobic barrier for preventing uncontrolled water loss and environmental damage. However, the mechanisms by which cuticle components are formed are still unknown in Brassica napus L. and were therefore assessed here. BnA1.CER4 and BnC1.CER4, encoding fatty acyl-coenzyme A reductases localizing to the endoplasmic reticulum and highly expressed in leaves, were identified and functionally characterized. Expression of BnA1.CER4 and BnC1.CER4 cDNA in yeast (Saccharomyces cerevisiae) induced the accumulation of primary alcohols with chain lengths of 26 carbons. The mutant line Nilla glossy2 exhibited reduced wax crystal types, and wax composition analysis showed that the levels of branched primary alcohols were decreased, whereas those of the other branched components were increased. Further analysis showed that the mutant had reduced water retention but enhanced resistance to Sclerotinia sclerotiorum. Collectively, our study reports that BnA1.CER4 and BnC1.CER4 are fatty acyl-coenzyme A reductase genes in B. napus with a preference for branched substrates that participate in the biosynthesis of anteiso-primary alcohols.

High-generation near-isogenic lines combined with multi-omics to study the mechanism of polima cytoplasmic male sterility.

BACKGROUND: Cytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima (pol) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the pol CMS restorer gene Rfp and the sterility-inducing gene orf224 in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration. RESULT: In this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene orf224 and the restorer gene Rfp of pol CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven Rfp interaction proteins, with orf224 protein covering five interaction proteins. CONCLUSIONS: We propose that Rfp and its interacting protein cleave the transcript of atp6/orf224, causing the infertility gene to lose its function and restore fertility. When Rfp is not cleaved, orf224 poisons the tapetum cells and anther development-related proteins, resulting in pol CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS's potential molecular mechanism and will help breed B. napus hybrids.

Exogenous Promoter Triggers APETALA3 Silencing through RNA-Directed DNA Methylation Pathway in Arabidopsis.

The development of floral organs plays a vital role in plant reproduction. In our research, the APETALA3 (AP3) promoter-transgenic lines showed abnormal developmental phenotypes in stamens and petals. The aim of this study is to understand the molecular mechanisms of the morphological defects in transgenic plants. By performing transgenic analysis, it was found that the AP3-promoted genes and the vector had no relation to the morphological defects. Then, we performed the expression analysis of the class A, B, and C genes. A dramatic reduction of transcript levels of class B genes (AP3 and PISTILLATA) was observed. Additionally, we also analyzed the methylation of the promoters of class B genes and found that the promoter of AP3 was hypermethylated. Furthermore, combining mutations in rdr2-2, drm1/2, and nrpd1b-11 with the AP3-silencing lines rescued the abnormal development of stamens and petals. The expression of AP3 was reactivated and the methylation level of AP3 promoter was also reduced in RdDM-defective AP3-silencing lines. Our results showed that the RdDM pathway contributed to the transcriptional silencing in the transgenic AP3-silencing lines. Moreover, the results revealed that fact that the exogenous fragment of a promoter could trigger the methylation of homologous endogenous sequences, which may be ubiquitous in transgenic plants.

Peri-mitral atrial flutter: personalized ablation strategy based on arrhythmogenic substrate.

Aims: The aim of this study is to characterize the arrhythmogenic substrate for peri-mitral atrial flutter (PMAFL), thereby determining a personalized ablation strategy to treat PMAFL. Methods and results: Thirty-six consecutive PMAFL patients (mean age: 63.8 ± 11.3, 23 males) underwent detailed three-dimensional electroanatomic mapping in left atrium (LA). The LA was divided into septal-anterior wall (SAW), posterior inferior wall (PIW), and mitral isthmus (MI) region, respectively. Ablation strategy was determined based on the endocardial bipolar voltage map. Based on electrophysiological substrates, 10, 17, and 9 cases were classified into iatrogenic, spontaneous, and no-substrate PMAFL, respectively. The mean voltage in SAW was significantly lower in spontaneous PMAFL (iatrogenic: 1.07 ± 0.66 mV; spontaneous: 0.65 ± 0.44 mV; no-substrate: 1.60 ± 0.53 mV, P <0.001), while iatrogenic PMAFL patients had the lowest voltage in MI (0.51 ± 0.23 mV vs. 1.55 ± 0.78 mV, 1.61 ± 0.56 mV, P <0.001). No low-voltage or slow conduction zone was found in the no-substrate PMAFL group. Fifteen spontaneous PMAFLs were successfully terminated by modified septal-anterior (9/10) or conventional anterior ablation line (6/7). Eight iatrogenic PMAFLs (8/10) were terminated by reinforcing the previous ablation areas. Cardioversion without PMAFL ablation was done in no-substrate PMAFL patients. After a median follow-up of 12 (7-39) months, two spontaneous PMAFL patients received redo procedures for recurrence due to "gap" conduction. Conclusions: The ablation strategy for PMAFL patients should be based on the arrhythmogenic substrate, but not the indiscriminate MI ablation. No-substrate PMAFLs during AF ablation could be monitored after cardioversion and might not need further ablation.

Genome-Wide Association Study Dissecting the Genetic Architecture Underlying the Branch Angle Trait in Rapeseed (Brassica napus L.).

The rapeseed branch angle is an important morphological trait because an adequate branch angle enables more efficient light capture under high planting densities. Here, we report that the average angle of the five top branches provides a reliable representation of the average angle of all branches. Statistical analyses revealed a significantly positive correlation between the branch angle and multiple plant-type and yield-related traits. The 60 K Brassica Infinium(®) single nucleotide polymorphism (SNP) array was utilized to genotype an association panel with 520 diverse accessions. A genome-wide association study was performed to determine the genetic architecture of branch angle, and 56 loci were identified as being significantly associated with the branch angle trait via three models, including a robust, novel, nonparametric Anderson-Darling (A-D) test. Moreover, these loci explained 51.1% of the phenotypic variation when a simple additive model was applied. Within the linkage disequilibrium (LD) decay ranges of 53 loci, we observed plausible candidates orthologous to documented Arabidopsis genes, such as LAZY1, SGR2, SGR4, SGR8, SGR9, PIN3, PIN7, CRK5, TIR1, and APD7. These results provide insight into the genetic basis of the branch angle trait in rapeseed and might facilitate marker-based breeding for improvements in plant architecture.

Genome-Wide Association Study Provides Insight into the Genetic Control of Plant Height in Rapeseed (Brassica napus L.).

Plant height is a key morphological trait of rapeseed. In this study, we measured plant height of a rapeseed population across six environments. This population contains 476 inbred lines representing the major Chinese rapeseed genepool and 44 lines from other countries. The 60K Brassica Infinium® SNP array was utilized to genotype the association panel. A genome-wide association study (GWAS) was performed via three methods, including a robust, novel, nonparametric Anderson-Darling (A-D) test. Consequently, 68 loci were identified as significantly associated with plant height (P < 5.22 × 10(-5)), and more than 70% of the loci (48) overlapped the confidence intervals of reported QTLs from nine mapping populations. Moreover, 24 GWAS loci were detected with selective sweep signals, which reflected the signatures of historical semi-dwarf breeding. In the linkage disequilibrium (LD) decay range up-and downstream of 65 loci (r (2) > 0.1), we found plausible candidates orthologous to the documented Arabidopsis genes involved in height regulation. One significant association found by GWAS colocalized with the established height locus BnRGA in rapeseed. Our results provide insights into the genetic basis of plant height in rapeseed and may facilitate marker-based breeding.

Catheter Ablation of Nonparoxysmal Atrial Fibrillation Using Electrophysiologically Guided Substrate Modification During Sinus Rhythm After Pulmonary Vein Isolation.

BACKGROUND: The high incidence of postprocedural atrial tachycardia reduces the absolute arrhythmia-free success rate of extensive ablation strategies to treat nonparoxysmal atrial fibrillation (NPAF). We hypothesized that a strategy of targeting low-voltage zones and sites with abnormal electrograms during sinus rhythm (SR-AEs) in the left atrium after circumferential pulmonary vein isolation and cavotricuspid isthmus ablation in patients with NPAF is superior. METHODS AND RESULTS: A total of 86 consecutive patients with NPAF were enrolled in study group. After circumferential pulmonary vein isolation, cavotricuspid isthmus ablation and cardioversion to SR, high-density mapping of left atrium was performed. Areas with low-voltage zone and SR-AE were targeted for further homogenization and elimination, respectively; 78 consecutive sex- and age-matched patients with NPAF who were treated with the stepwise approach served as the historical control group. In the study group, 92% (79/86) were successfully cardioverted after circumferential pulmonary vein isolation and cavotricuspid isthmus ablation. Among the patients converted to SR, 70% (55/79) had low-voltage zone and SR-AE and received additional ablation, whereas in 30% (24/79) without SR-AE or low-voltage zone, no further ablation was performed. During a follow-up period of >30 months, the Kaplan-Meier estimated probability to maintain SR at 24 months was 69.8% versus 51.3%. And after a single procedure, 3.5% (3/86) developed postprocedural atrial tachycardia in study group, compared with 30% (24/78) in control group (P=0.0003). CONCLUSIONS: A strategy of selective electrophysiologically guided atrial substrate modification in SR after circumferential pulmonary vein isolation and cavotricuspid isthmus ablation is clinically more effective than the stepwise approach for NPAF ablation. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov. Unique identifier: NCT01716143.

Comprehensive analysis of desmosomal gene mutations in Han Chinese patients with arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a cardiomyopathy that primarily involves the right ventricle. Mutations in desmosomal genes have been associated with ARVC. But its prevalence and spectrum are much less defined in the Chinese population, especially Han Chinese, a majority ethnic group in China; also the genotype-phenotype correlation regarding left ventricular involvement is still poorly understood. The aim of this study was to elucidate the genotype in Han Chinese patients with ARVC and the phenotype regarding cardiac left ventricle involvement in mutation carriers of ARVC. 48 Han Chinese patients were recruited into the present study based on the Original International Task Force Criteria of ARVC. Clinical data were reassessed according to the modified criteria published in 2010. A total of 36 subjects were diagnosed with ARVC; 12 patients were diagnosed with suspected ARVC. Five desmosomal genes (PKP2, DSG2, DSP, DSC2 and JUP) were sequenced directly from genomic DNA. Among the 36 patients, 21 mutations, 12 of which novel, were discovered in 19 individuals (19 of 36, 53%). The distribution of the mutations was 25% in PKP2, 14% in DSP, 11% in DSG2, 6% in JUP, and 3% in DSC2. Multiple mutations were identified in 2 subjects (2 of 36, 6%); both had digenic heterozygosity. Eight mutations, of which six were novel, were located in highly conserved regions. Seven mutations introduced a stop codon prematurely, which would result in premature termination of the protein synthesis. Two-dimensional echocardiography showed that LDVd and LDVs parameters were significantly larger in nonsense mutation carriers than in carriers of other mutations. In this comprehensive desmosome genetic analysis, 21 mutations were identified in five desmosomal genes in a group of 48 local Han Chinese subjects with ARVC, 12 of which were novel. PKP2 mutations were the most common variants. Left ventricular involvement could be a sign that the patient is a carrier of a nonsense cardiac desmosomal gene mutation.