The utility of whole-genome sequencing to inform epidemiologic investigations of SARS-CoV-2 clusters in acute-care hospitals.

OBJECTIVE: To evaluate the utility of selective reactive whole-genome sequencing (WGS) in aiding healthcare-associated cluster investigations. DESIGN: Mixed-methods quality-improvement study. SETTING: Thes study was conducted across 8 acute-care facilities in an integrated health system. METHODS: We analyzed healthcare-associated coronavirus disease 2019 (COVID-19) clusters between May 2020 and July 2022 for which facility infection prevention and control (IPC) teams selectively requested reactive WGS to aid the epidemiologic investigation. WGS was performed with real-time results provided to IPC teams, including genetic relatedness of sequenced isolates. We conducted structured interviews with IPC teams on the informativeness of WGS for transmission investigation and prevention. RESULTS: In total, 8 IPC teams requested WGS to aid the investigation of 17 COVID-19 clusters comprising 226 cases and 116 (51%) sequenced isolates. Of these, 16 (94%) clusters had at least 1 WGS-defined transmission event. IPC teams hypothesized transmission pathways in 14 (82%) of 17 clusters and used data visualizations to characterize these pathways in 11 clusters (65%). The teams reported that in 15 clusters (88%), WGS identified a transmission pathway; the WGS-defined pathway was not one that was predicted by epidemiologic investigation in 7 clusters (41%). WGS changed the understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in 8 clusters (47%) and altered infection prevention interventions in 8 clusters (47%). CONCLUSIONS: Selectively utilizing reactive WGS helped identify cryptic SARS-CoV-2 transmission pathways and frequently changed the understanding and response to SARS-CoV-2 outbreaks. Until WGS is widely adopted, a selective reactive WGS approach may be highly impactful in response to healthcare-associated cluster investigations.

Accurate and rapid prediction of tuberculosis drug resistance from genome sequence data using traditional machine learning algorithms and CNN.

Effective and timely antibiotic treatment depends on accurate and rapid in silico antimicrobial-resistant (AMR) predictions. Existing statistical rule-based Mycobacterium tuberculosis (MTB) drug resistance prediction methods using bacterial genomic sequencing data often achieve varying results: high accuracy on some antibiotics but relatively low accuracy on others. Traditional machine learning (ML) approaches have been applied to classify drug resistance for MTB and have shown more stable performance. However, there is no study that uses deep learning architecture like Convolutional Neural Network (CNN) on a large and diverse cohort of MTB samples for AMR prediction. We developed 24 binary classifiers of MTB drug resistance status across eight anti-MTB drugs and three different ML algorithms: logistic regression, random forest and 1D CNN using a training dataset of 10,575 MTB isolates collected from 16 countries across six continents, where an extended pan-genome reference was used for detecting genetic features. Our 1D CNN architecture was designed to integrate both sequential and non-sequential features. In terms of F1-scores, 1D CNN models are our best classifiers that are also more accurate and stable than the state-of-the-art rule-based tool Mykrobe predictor (81.1 to 93.8%, 93.7 to 96.2%, 93.1 to 94.8%, 95.9 to 97.2% and 97.1 to 98.2% for ethambutol, rifampicin, pyrazinamide, isoniazid and ofloxacin respectively). We applied filter-based feature selection to find AMR relevant features. All selected variant features are AMR-related ones in CARD database. 78.8% of them are also in the catalogue of MTB mutations that were recently identified as drug resistance-associated ones by WHO. To facilitate ML model development for AMR prediction, we packaged every step into an automated pipeline and shared the source code at https://github.com/KuangXY3/MTB-AMR-classification-CNN .

Evaluation of Five User-Friendly Whole Genome Sequencing Software for Mycobacterium tuberculosis in Clinical Application.

BACKGROUND: Whole genome sequencing (WGS) is an increasingly useful tool for tuberculosis (TB) diagnosis and disease management. In this study, we evaluated the utility of user-friendly WGS tools in reporting resistance profiles and identifying lineages of clinical TB isolates from South Korea. METHODS: Forty clinical samples from TB patients showing discrepancies between their rapid molecular and conventional drug susceptibility tests were used in this study. Among these clinical isolates, 37 strains were successfully evaluated via WGS software, using the GenTB, TB Profiler, PhyResSE, CASTB, and Mykrobe. RESULTS: More accurate and faster susceptibility results could be obtained with isoniazid than with rifampin. Using the phenotypic test as the gold standard, the isoniazid concordance rate between phenotypic drug susceptibility test (DST) and WGS (GenTB: 45.9%, TB profiler: 40.5%, PhyResSE: 40.5%, CASTB: 48.6%, and Mykrobe: 43.2%) was much higher than between phenotypic DST and rapid molecular genotypic DST (18.9%) among the 37 strains. In contrast, the rifampin concordance rate between phenotypic DST and WGS and that between phenotypic DST and rapid molecular genotypic DST was similar (81.1-89.2%). We also found novel mutations associated with INH in katG and ahpC gene region, not covered by the line probe assay. In addition, lineage analysis identified 81.1% of these samples as L2 East Asian lineage strains, and 18.9% as L4 Euro-American lineage strains. CONCLUSION: WGS may play a pivotal role in TB diagnosis and the detection of drug resistance, genetic diversity, and transmission dynamics in the near future because of its accuracy, speed, and extensibility.

Anti-osteosarcoma Biological Activity Evaluation and Complete Chloroplast Genome Sequencing of Populus yunnanensis.

Recently, the Populus yunnanensis extract has drawn the attention of most researchers, because of their anti-cancer activity. In this present research, the anti-cancer activity of the Populus yunnanensis extract was measured with Cell Counting Kit-8 (CCK-8) detection kit on the cancer cells. Then, the inhibitory activity of the Populus yunnanensis extract on the migration and invasion ability of the cancer cells was also determined in this present research with trans-well assay. Subsequently, to reveal the evolutionary genome evolution evaluation of the Populus yunnanensis and other Populus species, the high-throughput Illumina pair-end sequencing was performed and the chloroplast (cp) genome of Populus yunnanensis was determined, and the phylogenetic analysis was finished as wells. The results of the CCK-8 assay indicated that the Populus yunnanensis extract showed inhibitory effect on the cancer cell viability. Besides, the migration and invasion ability of the cancer cell was also reduced by the Populus yunnanensis extract. The complete chloroplast genome sequence results revealed that the Populus yunnanensis has a 156,505 bp circular cp genome. The phylogenetic analysis further revealed that the Populus yunnanensis has closely relationship with Populus simonii.

Genome-wide identification of the tea plant bHLH transcription factor family and discovery of candidate regulators of trichome formation.

Leaf trichomes play vital roles in plant resistance and the quality of tea. Basic helix-loop-helix (bHLH) transcription factors (TFs) play an important role in regulating plant development and growth. In this study, a total of 134 CsbHLH proteins were identified in the Camellia sinensis var. sinensis (CSS) genome. They were divided into 17 subgroups according to the Arabidopsis thaliana classification. Phylogenetic tree analysis indicated that members of subgroups IIIc-I and IIIc-II might be associated with trichome formation. The expression patterns of CsbHLH116, CsbHLH133, CsbHLH060, CsbHLH028, CsbHLH024, CsbHLH112 and CsbHLH053 from clusters 1, 3 and 5 were similar to the trichome distribution in tea plants. CsbHLH024 and CsbHLH133 were located in the cell nucleus and possessed transcriptional activation ability. They could interact with CsTTG1, which is a regulator of tea trichome formation. This study provides useful information for further research on the function of CsbHLHs in trichome formation.

Genome-wide analysis of nearly all the victims of a 6200 year old massacre.

Paleogenomic and bioanthropological studies of ancient massacres have highlighted sites where the victims were male and plausibly died all in battle, or were executed members of the same family as might be expected from a killing intentionally directed at subsets of a community, or where the massacred individuals were plausibly members of a migrant community in conflict with previously established groups, or where there was evidence that the killing was part of a religious ritual. Here we provide evidence of killing on a massive scale in prehistory that was not directed to a specific family, based on genome-wide ancient DNA for 38 of the 41 documented victims of a 6,200 year old massacre in Potocani, Croatia and combining our results with bioanthropological data. We highlight three results: (i) the majority of individuals were unrelated and instead were a sample of what was clearly a large farming population, (ii) the ancestry of the individuals was homogenous which makes it unlikely that the massacre was linked to the arrival of new genetic ancestry, and (iii) there were approximately equal numbers of males and females. Combined with the bioanthropological evidence that the victims were of a wide range of ages, these results show that large-scale indiscriminate killing is a horror that is not just a feature of the modern and historic periods, but was also a significant process in pre-state societies.

Real-World Utilization of Biomarker Testing for Patients with Advanced Non-Small Cell Lung Cancer in a Tertiary Referral Center and Referring Hospitals.

The continued introduction of biomarkers and innovative testing methods makes already complex diagnosis in patients with stage IV non-small-cell lung cancer (NSCLC) even more complex. This study primarily analyzed variations in biomarker testing in clinical practice in patients referred to a comprehensive cancer center in the Netherlands. The secondary aim was to compare the cost of biomarker testing with the cost of whole-genome sequencing. The cohort included 102 stage IV NSCLC patients who received biomarker testing in 2017 or 2018 at the comprehensive cancer center. The complete biomarker testing history of the cohort was identified using linked data from the comprehensive cancer center and the nationwide network and registry of histopathology and cytopathology in the Netherlands. Unique biomarker-test combinations, costs, turnaround times, and test utilization were examined. The results indicate substantial variation in test utilization and sequences. The mean cost per patient of biomarker testing was 2259.92 ± 1217.10 USD, or 1881.23 ± 1013.15 EUR. Targeted gene panels were most frequently conducted, followed by IHC analysis for programmed cell death protein ligand 1. Typically, the most common biomarkers were assessed within the first tests, and emerging biomarkers were tested further down the test sequence. At the cost of current biomarker testing, replacing current testing with whole-genome sequencing would have led to cost-savings in only two patients (2%).

Complete genome sequence of a novel foveavirus isolated from Allium sativum L. in China.

The complete genome sequence of a novel foveavirus identified in garlic (Allium sativum L.) in China was determined using RNA-seq, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The entire genomic RNA (GenBank accession MT981417) is 8748 nucleotides long excluding the 3'-terminal poly(A) tail and contains five open reading frames (ORFs). These ORFs encode the viral replicase, a triple gene block, and a coat protein. The virus was tentatively named "garlic yellow stripe associated virus" (GarYSaV). Pairwise comparisons of protein sequences show that GarYSaV encodes proteins that share less than 47% identity with those of other foveaviruses, suggesting that it represents a new species in the genus. Phylogenetic analysis of amino acid sequences of the replicase and CP confirm that GarYSaV is a member of the genus Foveavirus. To our knowledge, this is the first report of a foveavirus in a monocot plant.

Information, communication, and cancer patients' trust in the physician: what challenges do we have to face in an era of precision cancer medicine?

PURPOSE: Despite promising achievements in precision cancer medicine (PCM), participating patients are still faced with manifold uncertainties, especially regarding a potential treatment benefit of molecular diagnostics (MD). Hence, MD poses considerable challenges for patient information and communication. To meet these challenges, healthcare professionals need to gain deeper insight into patients' subjective experiences. Therefore, this qualitative study examined information aspects of MD programs in cancer patients. METHODS: In two German Comprehensive Cancer Centers, 30 cancer patients undergoing MD participated in semi-structured interviews on information transfer and information needs regarding MD. Additionally, patients provided sociodemographic and medical data and indicated their subjective level of information (visual analogue scale, VAS, 0-10). RESULTS: On average patients had high levels of information (mean = 7, median = 8); nevertheless 20% (n = 6) showed an information level below 5 points. Qualitative analysis revealed that patients show limited understanding of the complex background of MD and have uncertainties regarding their personal benefit. Further, patients described unmet information needs. Existential threat in awaiting the results was experienced as burdensome. To withstand the strains of their situation, patients emphasized the importance of trusting their physician. CONCLUSION: The challenges in PCM consist in providing unambiguous information, especially concerning treatment benefit, and providing guidance and support. Therefore, psycho-oncology needs to develop guidelines for adequate patient communication in order to help healthcare providers and cancer patients to handle these challenges in the developing field of PCM.

Analysis of Serial Multidrug-Resistant Tuberculosis Strains Causing Treatment Failure and Within-Host Evolution by Whole-Genome Sequencing.

The cure rate of multidrug-resistant tuberculosis (MDR-TB) is relatively low in China. The reasons for the treatment failure and within-host evolution during treatment have not been sufficiently studied. All MDR-TB patients receiving standard treatment from January 2014 to September 2016 at a designated TB Hospital in Zhejiang Province were retrospectively included and grouped according to their known treatment outcome. Clinical information was collected. Baseline strains of all patients and serial strains of treatment-failure patients were revived. Drug susceptibility tests (DSTs) of 14 drugs and single nucleotide polymorphism (SNP) analysis based on whole-genome sequencing (WGS) were performed. The genetic distance and within-host evolution were investigated based on SNPs. In total, 20 treatment failure patients and 74 patients who succeeded in treatment were included. The number of effective drugs for patients who failed treatment was no more than three. Eighteen (90.0%) treatment-failure patients were characterized by a continuous infection of the primary strain, of which 14 patients (77.8%) developed phenotypic or genotypic acquired drug resistance under ineffective treatment. Acquired resistance to amikacin and moxifloxacin (2.0 mg/ml) was detected most frequently, in 5 and 4 patients, respectively. The insufficient number of effective drugs in the combined treatment regimen was the main reason for MDR-TB treatment failure. The study emphasizes the importance of DST for second-line drugs when implementing the second-line drug regimen in MDR-TB patients. For patients with risk factors for MDR-TB, DST of second-line antituberculosis drugs should be performed at initiation of treatment. Second-line drugs should be selected based on the results of DST to avoid acquired resistance. WGS detects low-frequency resistance mutations and heterogeneous resistance with high sensitivity, which is of great significance for guiding clinical treatment and preventing acquired resistance.IMPORTANCE Few studies have focused on the reasons for the low cure rate of multidrug-resistant tuberculosis in China and within-host evolution during treatment, which is of great significance for improving clinical treatment regimens. Acquired resistance events were common during the ineffective treatment, among which resistance to amikacin and high-level moxifloxacin were the most common. The main reason for the treatment failure of MDR-TB patients was insufficient effective drugs, which may lead to higher levels of drug resistance in MDR-TB strains. Therefore, the study emphasizes the importance of DST in the development of second-line treatment regimen when there is a risk of MDR. By performing whole-genome sequencing of serial strains from patients with treatment failure, we found that WGS can detect low-frequency resistance mutations and heterogeneous resistance with high sensitivity. It is thus recommended to conduct drug susceptibility tests at the beginning of treatment and repeat the DST when the sputum bacteria remain positive.

Study protocol: Whole genome sequencing Implementation in standard Diagnostics for Every cancer patient (WIDE).

BACKGROUND: 'Precision oncology' can ensure the best suitable treatment at the right time by tailoring treatment towards individual patient and comprehensive tumour characteristics. In current molecular pathology, diagnostic tests which are part of the standard of care (SOC) only cover a limited part of the spectrum of genomic changes, and often are performed in an iterative way. This occurs at the expense of valuable patient time, available tissue sample, and interferes with 'first time right' treatment decisions. Whole Genome Sequencing (WGS) captures a near complete view of genomic characteristics of a tumour in a single test. Moreover, WGS facilitates faster implementation of new treatment relevant biomarkers. At present, WGS mainly has been applied in study settings, but its performance in a routine diagnostic setting remains to be evaluated. The WIDE study aims to investigate the feasibility and validity of WGS-based diagnostics in clinical practice. METHODS: 1200 consecutive patients in a single comprehensive cancer centre with (suspicion of) a metastasized solid tumour will be enrolled with the intention to analyse tumour tissue with WGS, in parallel to SOC diagnostics. Primary endpoints are (1) feasibility of implementation of WGS-based diagnostics into routine clinical care and (2) clinical validation of WGS by comparing identification of treatment-relevant variants between WGS and SOC molecular diagnostics. Secondary endpoints entail (1) added clinical value in terms of additional treatment options and (2) cost-effectiveness of WGS compared to SOC diagnostics through a Health Technology Assessment (HTA) analysis. Furthermore, the (3) perceived impact of WGS-based diagnostics on clinical decision making will be evaluated through questionnaires. The number of patients included in (experimental) therapies initiated based on SOC or WGS diagnostics will be reported with at least 3 months follow-up. The clinical efficacy is beyond the scope of WIDE. Key performance indicators will be evaluated after every 200 patients enrolled, and procedures optimized accordingly, to continuously improve the diagnostic performance of WGS in a routine clinical setting. DISCUSSION: WIDE will yield the optimal conditions under which WGS can be implemented in a routine molecular diagnostics setting and establish the position of WGS compared to SOC diagnostics in routine clinical care.

Complete genome sequence of Sphingobium sp. strain PAMC 28499 reveals a potential for degrading pectin with comparative genomics approach.

BACKGROUND: Spingobium sp. PAMC 28499 is isolated from the glaciers of Uganda. Uganda is a unique region where hot areas and glaciers coexist, with a variety of living creatures surviving, but the survey on them is very poor. The genetic character and complete genome information of Sphingobium strains help with environmental studies and the development of better to enzyme industry. OBJECTIVE: In this study, complete genome sequence of Spingobium sp. PAMC 28499 and comparative analysis of Spingobium species strains isolated from variety of the region. METHODS: Genome sequencing was performed using PacBio sequel single-molecule real-time (SMRT) sequencing technology. The predicted gene sequences were functionally annotated and gene prediction was carried out using the program NCBI non-redundant database. And using dbCAN2 and KEGG data base were degradation pathway predicted and protein prediction about carbohydrate active enzymes (CAZymes). RESULTS: The genome sequence has 64.5% GC content, 4432 coding protein coding genes, 61 tRNAs, and 12 rRNA operons. Its genome encodes a simple set of metabolic pathways relevant to pectin and its predicted degradation protein an unusual distribution of CAZymes with extracellular esterases and pectate lyases. CAZyme annotation analyses revealed 165 genes related to carbohydrate active, and especially we have found GH1, GH2, GH3, GH38, GH35, GH51, GH51, GH53, GH106, GH146, CE12, PL1 and PL11 such as known pectin degradation genes from Sphingobium yanoikuiae. These results confirmed that this Sphingobium sp. strain PAMC 28499 have similar patterns to RG I pectin-degrading pathway. CONCLUSION: In this study, isolated and sequenced the complete genome of Spingobium sp. PAMC 28499. Also, this strain has comparative genome analysis. Through the complete genome we can predict how this strain can store and produce energy in extreme environment. It can also provide bioengineered data by finding new genes that degradation the pectin.

Characterization and Comparative Analysis of Two Rheum Complete Chloroplast Genomes.

Rheum species present a significant economic value. Traditional Chinese medicine rhubarb is an important medicinal material in China. It has a long history of use, with a record of use as early as two thousand years ago. Here, we determined the complete chloroplast genome sequences of Rheum nobile and Rheum acuminatum and comprehensively compared them to two other available Rheum cp genomes at the genome scale. The results revealed cp genomes ranging in size from 159,051 to 161,707 bp with a similar typical quadripartite and circular structure. The genome organization, gene numbers, gene order, and GC contents of these four Rheum cp genomes were similar to those of many angiosperm cp genomes. Repeats and microsatellites were detected in the R. nobile and R. acuminatum cp genomes. The Mauve alignment revealed that there were no rearrangements in the cp genomes of the four Rheum species. Thirteen mutational hotspots for genome divergence were identified, which could be utilized as potential markers for phylogenetic studies and the identification of Rheum species. The phylogenetic relationships of the four species showed that the members of Rheum cluster into a single clade, indicating their close relationships. Our study provides valuable information for the taxonomic, phylogenetic, and evolutionary analysis of Rheum.

Characterization and improvement of curdlan produced by a high-yield mutant of Agrobacterium sp. ATCC 31749 based on whole-genome analysis.

Curdlan is a bacterial, water-insoluble, linear homopolysaccharide that has been widely used in the food industry. In this study, genome information of strain CGMCC 11546, a UV-induced high-yield mutant of the model curdlan-producing strain Agrobacterium sp. ATCC 31749, was used to investigate the molecular mechanism of curdlan biosynthesis. The maximum curdlan yield of 47.97 ± 0.57 g/L was obtained from strain CGMCC 11546 by using optimal media containing 60 g/L sucrose, 6 g/L yeast, 2 g/L KH2PO4, 0.4 g/L MgSO4·7H2O, 2 g/L CaCO3, 0.1 g/L FeSO4·7H2O, 0.04 g/L MnSO4, and 0.02 g/L ZnCl2 at 30 °C and 280 rpm after 96 h of fermentation. The gel strength of curdlan was improved by 41 % by knocking out the ß-1,3-glucanase genes exoK and exsH of strain CGMCC 11546. Furthermore, the application of curdlan from the ΔexoK-exsH strain in noodles significantly improved the eating quality of both raw and cooked noodles.

Genetic dissection of an allotetraploid interspecific CSSLs guides interspecific genetics and breeding in cotton.

BACKGROUND: The low genetic diversity of Upland cotton limits the potential for genetic improvement. Making full use of the genetic resources of Sea-island cotton will facilitate genetic improvement of widely cultivated Upland cotton varieties. The chromosome segments substitution lines (CSSLs) provide an ideal strategy for mapping quantitative trait loci (QTL) in interspecific hybridization. RESULTS: In this study, a CSSL population was developed by PCR-based markers assisted selection (MAS), derived from the crossing and backcrossing of Gossypium hirsutum (Gh) and G. barbadense (Gb), firstly. Then, by whole genome re-sequencing, 11,653,661 high-quality single nucleotide polymorphisms (SNPs) were identified which ultimately constructed 1211 recombination chromosome introgression segments from Gb. The sequencing-based physical map provided more accurate introgressions than the PCR-based markers. By exploiting CSSLs with mutant morphological traits, the genes responding for leaf shape and fuzz-less mutation in the Gb were identified. Based on a high-resolution recombination bin map to uncover genetic loci determining the phenotypic variance between Gh and Gb, 64 QTLs were identified for 14 agronomic traits with an interval length of 158 kb to 27 Mb. Surprisingly, multiple alleles of Gb showed extremely high value in enhancing cottonseed oil content (SOC). CONCLUSIONS: This study provides guidance for studying interspecific inheritance, especially breeding researchers, for future studies using the traditional PCR-based molecular markers and high-throughput re-sequencing technology in the study of CSSLs. Available resources include candidate position for controlling cotton quality and quantitative traits, and excellent breeding materials. Collectively, our results provide insights into the genetic effects of Gb alleles on the Gh, and provide guidance for the utilization of Gb alleles in interspecific breeding.

Whole-genome sequencing analysis of multidrug-resistant Mycobacterium tuberculosis from Java, Indonesia.

Introduction. Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem globally, including in Indonesia. Whole-genome sequencing (WGS) analysis has rarely been used for the study of TB and MDR-TB in Indonesia.Aim. We evaluated the use of WGS for drug-susceptibility testing (DST) and to investigate the population structure of drug-resistant Mycobacterium tuberculosis in Java, Indonesia.Methodology. Thirty suspected MDR-TB isolates were subjected to MGIT 960 system (MGIT)-based DST and to WGS. Phylogenetic analysis was done using the WGS data. Results obtained using MGIT-based DST and WGS-based DST were compared.Results. Agreement between WGS and MGIT was 93.33 % for rifampicin, 83.33 % for isoniazid and 76.67 % for streptomycin but only 63.33 % for ethambutol. Moderate WGS-MGIT agreement was found for second-line drugs including amikacin, kanamycin and fluoroquinolone (73.33-76.67 %). MDR-TB was more common in isolates of the East Asian Lineage (63.3%). No evidence of clonal transmission of DR-TB was found among members of the tested population.Conclusion. Our study demonstrated the applicability of WGS for DST and molecular epidemiology of DR-TB in Java, Indonesia. We found no transmission of DR-TB in Indonesia.

Comparative chloroplast genome analysis of Artemisia (Asteraceae) in East Asia: insights into evolutionary divergence and phylogenomic implications.

BACKGROUND: Artemisia in East Asia includes a number of economically important taxa that are widely used for food, medicinal, and ornamental purposes. The identification of taxa, however, has been hampered by insufficient diagnostic morphological characteristics and frequent natural hybridization. Development of novel DNA markers or barcodes with sufficient resolution to resolve taxonomic issues of Artemisia in East Asia is significant challenge. RESULTS: To establish a molecular basis for taxonomic identification and comparative phylogenomic analysis of Artemisia, we newly determined 19 chloroplast genome (plastome) sequences of 18 Artemisia taxa in East Asia, de novo-assembled and annotated the plastomes of two taxa using publicly available Illumina reads, and compared them with 11 Artemisia plastomes reported previously. The plastomes of Artemisia were 150,858-151,318 base pairs (bp) in length and harbored 87 protein-coding genes, 37 transfer RNAs, and 8 ribosomal RNA genes in conserved order and orientation. Evolutionary analyses of whole plastomes and 80 non-redundant protein-coding genes revealed that the noncoding trnH-psbA spacer was highly variable in size and nucleotide sequence both between and within taxa, whereas the coding sequences of accD and ycf1 were under weak positive selection and relaxed selective constraints, respectively. Phylogenetic analysis of the whole plastomes based on maximum likelihood and Bayesian inference analyses yielded five groups of Artemisia plastomes clustered in the monophyletic subgenus Dracunculus and paraphyletic subgenus Artemisia, suggesting that the whole plastomes can be used as molecular markers to infer the chloroplast haplotypes of Artemisia taxa. Additionally, analysis of accD and ycf1 hotspots enabled the development of novel markers potentially applicable across the family Asteraceae with high discriminatory power. CONCLUSIONS: The complete sequences of the Artemisia plastomes are sufficiently polymorphic to be used as super-barcodes for this genus. It will facilitate the development of new molecular markers and study of the phylogenomic relationships of Artemisia species in the family Asteraceae.

Identification and characterization of the stunted sterile (ss) mutant in rice.

BACKGROUND: Proper organ development is pivotal for normal rice growth and production. Many genes are involved in this process, and these genes provide a basis for rice breeding. OBJECTIVE: To identify a novel mutation causing developmental defects in rice. METHODS: The phenotype of a rice mutant, stunted sterile (ss), identified from the japonica rice cultivar Samkwang treated with N-methyl-N-nitrosourea, was characterized, including anatomical and pollen activity analyses. A genetic analysis and fine mapping were performed to identify a candidate locus, followed by a sequence analysis to determine the causal mutation for the phenotype. RESULTS: Compared with wild-type plants, the mutant exhibited a 34% reduction in height, 46% reduction in flag leaf width, and complete panicle sterility. Cell proliferation in the leaf and pollen viability were significantly inhibited in the mutant. The mutant phenotypes were controlled by a single recessive gene that was fine-mapped to an 84 kb region between two SNP markers on the short arm of chromosome 5. A candidate gene analysis determined that the mutant carries an 11 bp insertion in the coding region of LOC_Os05g03550, which encodes a protein containing two SANT domains, resulting in a premature termination codon before the conserved domain. CONCLUSIONS: We identified a novel rice gene, Stunted sterile, involved in the regulation of various developmental processes. Our findings improve our understanding of the role of chromatin remodeling in organ development and have implications for breeding owing to the broad effects of the gene on plant growth.

Genome-wide assessment of population structure and genetic diversity of Chinese Lou onion using specific length amplified fragment (SLAF) sequencing.

Lou onion (Allium fistulosum L. var. viviparum) is an abundant source of flavonols which provides additional health benefits to diseases. Genome-wide specific length amplified fragment (SLAF) sequencing method is a rapidly developed deep sequencing technologies used for selection and identification of genetic loci or markers. This study aimed to elucidate the genetic diversity of 122 onion accessions in China using the SLAF-seq method. A set of 122 onion accessions including 107 A.fistulosum L. var. viviparum Makino, 3 A.fistulosum L. var. gigantum Makino, 3 A.mongolicum Regel and 9 A.cepa L. accessions (3 whites, 3 reds and 3 yellows) from different regions in China were enrolled. Genomic DNA was isolated from young leaves and prepared for the SLAF-seq, which generated a total of 1,387.55 M reads and 162,321 high quality SNPs (integrity >0.5 and MAF >0.05). These SNPs were used for the construction of neighbor-joining phylogenetic tree, in which 10 A.fistulosum L. var. viviparum Makino accessions from Yinchuan (Ningxia province) and Datong (Qinghai province) had close genetic relationship. The 3 A.cepa L. clusters (red, white and yellow) had close genetic relationship especially with the 97 A.fistulosum L. var. viviparum Makino accessions. Population structure analysis suggested entire population could be clustered into 3 groups, while principal component analysis (PCA) showed there were 4 genetic groups. We confirmed the SLAF-seq approach was effective in genetic diversity analysis in red onion accessions. The key findings would provide a reference to the Lou onion germplasm in China.

Genome-wide identification and characterization of microRNAs by small RNA sequencing for low nitrogen stress in potato.

Nitrogen is an important nutrient for plant growth and tuber quality of potato. Since potato crop requires high dose of N, improving nitrogen use efficiency (NUE) of plant is an inevitable approach to minimize N fertilization. The aim of this study was to identify and characterize microRNAs (miRNAs) by small RNA sequencing in potato plants grown in aeroponic under two contrasting N (high and low) regimes. A total of 119 conserved miRNAs belonging to 41 miRNAs families, and 1002 putative novel miRNAs were identified. From total, 52 and 54 conserved miRNAs, and 404 and 628 putative novel miRNAs were differentially expressed in roots and shoots, respectively under low N stress. Of total 34,135 predicted targets, the gene ontology (GO) analysis indicated that maximum targets belong to biological process followed by molecular function and cellular component. Eexpression levels of the selected miRNAs and targets were validated by real time-quantitative polymerase chain reaction (RT-qPCR) analysis. Two predicted targets of potential miRNAs (miR397 and miR398) were validated by 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). In general, predicted targets are associated with stress-related, kinase, transporters and transcription factors such as universal stress protein, heat shock protein, salt-tolerance protein, calmodulin binding protein, serine-threonine protein kinsae, Cdk10/11- cyclin dependent kinase, amino acid transporter, nitrate transporter, sugar transporter, transcription factor, F-box family protein, and zinc finger protein etc. Our study highlights that miR397 and miR398 play crucial role in potato during low N stress management. Moreover, study provides insights to modulate miRNAs and their predicted targets to develop N-use efficient potato using transgenic/genome-editing tools in future.