Creating a zero amylose barley with high soluble sugar content by genome editing.

Amylose biosynthesis is strictly associated with granule-bound starch synthase I (GBSSI) encoded by the Waxy gene. Mutagenesis of single bases in the Waxy gene, which induced by CRISPR/Cas9 genome editing, caused absence of intact GBSSI protein in grain of the edited line. The amylose and amylopectin contents of waxy mutants were zero and 31.73%, while those in the wild type were 33.50% and 39.00%, respectively. The absence of GBSSI protein led to increase in soluble sugar content to 37.30% compared with only 10.0% in the wild type. Sucrose and ß-glucan, were 39.16% and 35.40% higher in waxy mutants than in the wild type, respectively. Transcriptome analysis identified differences between the wild type and waxy mutants that could partly explain the reduction in amylose and amylopectin contents and the increase in soluble sugar, sucrose and ß-glucan contents. This waxy flour, which showed lower final viscosity and setback, and higher breakdown, could provide more option for food processing.

Application and diagnostic points of carotid artery ultrasound in the detection of internal carotid artery absence: Analysis of 16 cases and literature review.

OBJECTIVE: To summarize the ultrasound diagnostic key points and imaging characteristics of absence of the internal carotid artery (ICA), and to explore the application value of carotid artery ultrasound in the diagnosis of ICA absence. METHODS: Sixteen patients diagnosed with ICA absence at our hospital were retrospectively analyzed. Clinical data, ultrasound images, and other relevant imaging data were reviewed. The ultrasound diagnostic key points and related imaging characteristics of ICA absence were summarized. Combined with a literature review, the application of carotid artery ultrasound in the diagnosis of ICA absence was discussed. RESULTS: Among the 16 patients, there were 10 males and 6 females ages ranging from 13 to 80 years. Among them, 5 patients had a history of cerebral infarction, 1 patient had a history of subarachnoid hemorrhage, and 10 patients had no history of stroke. Among the 16 patients, there were 10 left-sided lesions, 5 right-sided lesions, and 1 bilateral lesion. Three patients had concomitant aneurysms. Carotid ultrasound examinations in all 16 ICA absent patients revealed that a significantly slender common carotid artery (CCA) diameter compared to the contralateral (3.91 ± 0.73 vs. 6.92 ± 1.42, p < 0.001). On the side of ICA absence, the bifurcation of the carotid artery was not detected, with the CCA directly continuing as a single artery (external carotid artery, ECA). The temporal superficial artery percussion test was positive in all cases, and the blood flow spectrum of the single artery (ECA) on the side of the absent ICA showed a serrated pattern during diastole. On the side where the ICA was absent, the blood flow spectrum morphology of the CCA and ECA was consistent. Based on other relevant imaging examinations, the patients were classified into Lie types: A (10 cases), B (2 cases), C (1 case), D (1 case), and 2 cases were unclassified. CONCLUSION: Carotid artery ultrasound has the advantages of easy operation, affordability, non-invasiveness, and capable of dynamic and high repeatability. Compared to other imaging examinations, it can provide more evidence for the diagnosis of ICA absence, and is suitable for widespread application and promotion.

Induce male sterility by CRISPR/Cas9-mediated mitochondrial genome editing in tobacco.

Genome editing has become more and more popular in animal and plant systems following the emergence of CRISPR/Cas9 technology. However, target sequence modification by CRISPR/Cas9 has not been reported in the plant mitochondrial genome, mtDNA. In plants, a type of male sterility known as cytoplasmic male sterility (CMS) has been associated with certain mitochondrial genes, but few genes have been confirmed by direct mitochondrial gene-targeted modifications. Here, the CMS-associated gene (mtatp9) in tobacco was cleaved using mitoCRISPR/Cas9 with a mitochondrial localization signal. The male-sterile mutant, with aborted stamens, exhibited only 70% of the mtDNA copy number of the wild type and exhibited an altered percentage of heteroplasmic mtatp9 alleles; otherwise, the seed setting rate of the mutant flowers was zero. Transcriptomic analyses showed that glycolysis, tricarboxylic acid cycle metabolism and the oxidative phosphorylation pathway, which are all related to aerobic respiration, were inhibited in stamens of the male-sterile gene-edited mutant. In addition, overexpression of the synonymous mutations dsmtatp9 could restore fertility to the male-sterile mutant. Our results strongly suggest that mutation of mtatp9 causes CMS and that mitoCRISPR/Cas9 can be used to modify the mitochondrial genome of plants.

Critical contributions of protein cargos to the functions of macrophage-derived extracellular vesicles.

BACKGROUND: Macrophages are highly plastic innate immune cells that play key roles in host defense, tissue repair, and homeostasis maintenance. In response to divergent stimuli, macrophages rapidly alter their functions and manifest a wide polarization spectrum with two extremes: M1 or classical activation and M2 or alternative activation. Extracellular vesicles (EVs) secreted from differentially activated macrophages have been shown to have diverse functions, which are primarily attributed to their microRNA cargos. The role of protein cargos in these EVs remains largely unexplored. Therefore, in this study, we focused on the protein cargos in macrophage-derived EVs. RESULTS: Naïve murine bone marrow-derived macrophages were treated with lipopolysaccharide or interlukin-4 to induce M1 or M2 macrophages, respectively. The proteins of EVs and their parental macrophages were subjected to quantitative proteomics analyses, followed by bioinformatic analyses. The enriched proteins of M1-EVs were involved in proinflammatory pathways and those of M2-EVs were associated with immunomodulation and tissue remodeling. The signature proteins of EVs shared a limited subset of the proteins of their respective progenitor macrophages, but they covered many of the typical pathways and functions of their parental cells, suggesting their respective M1-like and M2-like phenotypes and functions. Experimental examination validated that protein cargos in M1- or M2-EVs induced M1 or M2 polarization, respectively. More importantly, proteins in M1-EVs promoted viability, proliferation, and activation of T lymphocytes, whereas proteins in M2-EVs potently protected the tight junction structure and barrier integrity of epithelial cells from disruption. Intravenous administration of M2-EVs in colitis mice led to their accumulation in the colon, alleviation of colonic inflammation, promotion of M2 macrophage polarization, and improvement of gut barrier functions. Protein cargos in M2-EVs played a key role in their protective function in colitis. CONCLUSION: This study has yielded a comprehensive unbiased dataset of protein cargos in macrophage-derived EVs, provided a systemic view of their potential functions, and highlighted the important engagement of protein cargos in the pathophysiological functions of these EVs.

Functional R2R3-MYB transcription factor NsMYB1, regulating anthocyanin biosynthesis, was relative to the fruit color differentiation in Nitraria sibirica Pall.

BACKGROUND: Nitraria sibirica Pall. is an economic plant with two kinds of fruit color, widely spreads in the Qinghai Tibet Plateau. The chemical analysis and pharmacological evaluation had been carried out for several tens of years, the mechanism behind the fruit color differentiation is still unclear. RESULTS: In this manuscript, the chemical analysis of the extractions showed that the chemical composition of fruit color was anthocyanin, and two kind of Nitraria sibirica Pall. were caused by the content differentiation with the same anthocyanin kinds. Cyanidin-3-[2"-(6'"-coumaroyl)-glucosyl]-glucoside (C3G) was the major anthocyanin. Transcriptome analysis and the qRT-PCR revealed that the structural genes relative to anthocyanin biosynthesis except CHS, F3'5'H and ANS were up-regulated in the peels of BF (Black fruit) compared with the peels of RF (Red fruit), which indicated that transcript factor should be the reason for the expression difference of the structure genes. In the unigenes of the transcript factor MYB and bHLH, relative to anthocyanin, only NsMYB1 (Cluster 8422.10600), was high-expression and up-expression in the peels of BF. NsMYB1 encoded the same length protein with four amino acid differences in the RF and BF, and both contained the intact DNA, HTH-MYB and SANT domains. NsMYB1 was close to the AtMYB114, AtMYB113 and AtPAP1, regulating anthocyanin biosynthesis, in phylogenetic relationship. Both NsMYB1r and NsMYB1b could promote the transcript of the structural genes, and induced the anthocyanin accumulation in all tissues of transgenic tobacco. The insertion of 'TATA' in the promoter of NsMYB1r gave one more promoter region, and was the reason for higher transcripts in black fruit possibly. CONCLUSIONS: Cyanidin-3-[2''-(6'"-coumaroyl)-glucosyl]-glucoside was the major anthocyanin in black fruit of Nitraria sibirica Pall.. NsMYB1 was a functional R2R3-MYB transcription factor, regulated the anthocyanin biosynthesis, and led to the fruit color differentiation in Nitraria sibirica Pall.

Genotyping-by-sequencing and genome-wide association study reveal genetic diversity and loci controlling agronomic traits in triticale.

KEY MESSAGE: The genetic diversity and loci underlying agronomic traits were analyzed by the reads coverage and genome-wide association study based genotyping-by-sequencing in a diverse population consisting of 199 accessions. Triticale (× Triticosecale Wittmack) is an economically important grain forage and energy crop planted worldwide for its high biomass. Little is known about the genetic diversity and loci underlying agronomic traits in triticale. We performed genotyping-by-sequencing of 199 cultivars and mapped reads to the A, B, D, and R genomes for karyotype analysis. These cultivars could mostly be grouped into five types. Some chromosome abnormalities occurred with high frequency, such as 2D (2R) substitution, deletion of the long arm of chromosome 2D or the short arm of 5R, and translocation of the long arms of 7D/7A, the short arms of 6D/6A, or the long arms of 1D/1A. We chose only widely planted hexaploid triticale cultivars (153) for genome-wide association study. These cultivars could be divided into nine distinct groups, and the linkage disequilibrium decay was 25.4 kb in this population. We identified 253 significant marker-trait associations (MTAs) on 20 chromosomes, except 7R. Twenty-one reliable MTAs were identified repeatedly over two environments. We predicted 16 putative candidate genes involved in plant growth and development using the genome sequences of wheat and rye. These results provide a basis for understanding the genetic mechanisms of agronomic traits and will benefit the breeding of improved hexaploid triticale.

Novel Hina alleles created by genome editing increase grain hardness and reduce grain width in barley.

The hordoindolina genes (Hina and Hinb) are believed to play critical roles in barley (Hordeum vulgare L.) grain texture. In this study, we created novel alleles of the Hina gene using CRISPR/Cas9 (Clustered regularly inter spaced short palindromic repeat-associated protein, CRISPR-Cas) genome editing. Mutagenesis of single bases in these novel alleles led to loss of Hina protein function in edited lines. The grain hardness index of hina mutants was 95.5 on average, while that of the wild type was only 53.7, indicating successful conversion of soft barley into hard barley. Observation of cross-sectional grain structure using scanning electron microscopy revealed different adhesion levels between starch granules and protein matrix. Starch granules were loose and separated from the protein matrix in the wild type, but deeply trapped and tightly integrated with the protein matrix in hina02 mutants. In addition, the grain width and thousand-grain weight of the hina02 mutant were significantly lower than those of the wild type.

The value of color Doppler sonography in diagnosis and hemodynamic analysis of innominate artery occlusion.

The occlusion of the innominate artery caused a significant decrease in the distal end of the right subclavian artery and the right common carotid artery, internal carotid artery, and external carotid artery (ECA). Due to the different pressure and the abundant communicating arteries between the ECA and the bilateral vertebral artery (VA), different paths of blood steal in the anterior and posterior circulation occurred.

De novo genome assembly of a foxtail millet cultivar Huagu11 uncovered the genetic difference to the cultivar Yugu1, and the genetic mechanism of imazethapyr tolerance.

BACKGROUND: Setaria italica is the second-most widely planted species of millets in the world and an important model grain crop for the research of C4 photosynthesis and abiotic stress tolerance. Through three genomes assembly and annotation efforts, all genomes were based on next generation sequencing technology, which limited the genome continuity. RESULTS: Here we report a high-quality whole-genome of new cultivar Huagu11, using single-molecule real-time sequencing and High-throughput chromosome conformation capture (Hi-C) mapping technologies. The total assembly size of the Huagu11 genome was 408.37 Mb with a scaffold N50 size of 45.89 Mb. Compared with the other three reported millet genomes based on the next generation sequencing technology, the Huagu11 genome had the highest genomic continuity. Intraspecies comparison showed about 94.97 and 94.66% of the Yugu1 and Huagu11 genomes, respectively, were able to be aligned as one-to-one blocks with four chromosome inversion. The Huagu11 genome contained approximately 19.43 Mb Presence/absence Variation (PAV) with 627 protein-coding transcripts, while Yugu1 genomes had 20.53 Mb PAV sequences encoding 737 proteins. Overall, 969,596 Single-nucleotide polymorphism (SNPs) and 156,282 insertion-deletion (InDels) were identified between these two genomes. The genome comparison between Huagu11 and Yugu1 should reflect the genetic identity and variation between the cultivars of foxtail millet to a certain extent. The Ser-626-Aln substitution in acetohydroxy acid synthase (AHAS) was found to be relative to the imazethapyr tolerance in Huagu11. CONCLUSIONS: A new improved high-quality reference genome sequence of Setaria italica was assembled, and intraspecies genome comparison determined the genetic identity and variation between the cultivars of foxtail millet. Based on the genome sequence, it was inferred that the Ser-626-Aln substitution in AHAS was responsible for the imazethapyr tolerance in Huagu11. The new improved reference genome of Setaria italica will promote the genic and genomic studies of this species and be beneficial for cultivar improvement.

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L.

BACKGROUND: Tuber color is an important trait for Helianthus tuberosus L. (Jerusalem artichoke). Usually, purple tubers with high anthocyanin content are more nutritious than white tuber. But, the molecular mechanism underlying it is unknown. RESULTS: In the current study, high-throughput RNA-sequencing was used to compare the transcriptomes between plants with tubers with red or white epidermis. Compared with the white-skinned tubers of cultivar QY3, anthocyanin biosynthesis structural genes had greater expression in the red-skinned tubers of cultivar QY1, indicating that the anthocyanin biosynthesis pathway was activated in 'QY1'; quantitative PCR confirmed this difference in expression. HtMYB2 (Unigene44371_All) was the only MYB transcription factor, homologous to the MYB transcription factor regulating anthocyanin biosynthesis, expressed in the red tuber epidermis of 'QY1'. The anthocyanin concentration in the root, stem, leaf, flower, and tuber epidermis of 'QY1' was higher than in 'QY3', especially tuber epidermis. Correspondingly, HtMYB2 had greater expression in these tissues of 'QY1' than in 'QY3'. The expression of HtMYB2 was associated with anthocyanin accumulation in the different tissues. Overexpression of HtMYB2 activated the anthocyanin biosynthesis pathway, accumulating the pigment in leaves of transgenic tobacco, supporting the model that HtMYB2 regulated anthocyanin biosynthesis. Further experiments found that HtMYB2 had the same coding sequence and genomic sequence in 'QY1' and 'QY3', but that there were several single nucleotide polymorphisms and one insertion-deletion (indel) mutation of 21 nucleotides in the promoter region between the two alleles. The deletion of three nucleotides "AAA" made the promoter of 'QY1' predicted to contain one more possible promoter region. A specific primer, based on the indel, could differentiate between cultivars with red or white tuber epidermis. The genetic variation in HtMYB2 was associated with the tuber skin color in a natural population. CONCLUSIONS: RNA-seq can successfully isolate the candidate gene (HTMYB2) controlling anthocyanin biosynthesis in purple epidermis of Jerusalem artichoke tuber. HTMYB2 can regulate anthocyanin biosynthesis in plants and is closely related to the formation of purple phenotype in tubers. This study should be useful in understanding the genetic mechanism underlying different tuber skin colors and in breeding new H. tuberosus cultivars with different tuber skin colors.