The occurrence,inheritance,and segregation of complex genomic structural variation in synthetic Brassica napus

"Synthetic"allopolyploids recreated by interspecific hybridization play an important role in providing novel genomic variation for crop improvement.Such synthetic allopolyploids often undergo rapid genomic structural variation(SV).However,how such SV arises,is inherited and fixed,and how it affects important traits,has rarely been comprehensively and quantitively studied in advanced generation synthetic lines.A better understanding of these processes will aid breeders in knowing how to best utilize synthetic allopolyploids in breeding programs.Here,we analyzed three genetic mapping populations(735 DH lines)derived from crosses between advanced synthetic and conventional Brassica napus(rapeseed)lines,using whole-genome sequencing to determine genome composition.We observed high tolerance of large structural variants,particularly toward the telomeres,and preferential selection for balanced homoeologous exchanges(duplication/deletion events between the A and C genomes resulting in retention of gene/chromosome dosage between homoeologous chromosome pairs),including stable events involving whole chromosomes("pseudoeuploidy").Given the experimental design(all three populations shared a common parent),we were able to observe that parental SV was regularly inherited,showed genetic hitchhiking effects on segregation,and was one of the major factors inducing adjacent novel and larger SV.Surprisingly,novel SV occurred at low frequencies with no significant impacts on observed fertility and yield-related traits in the advanced generation synthetic lines.However,incorporating genome-wide SV in linkage mapping explained significantly more genetic variance for traits.Our results provide a framework for detecting and understanding the occurrence and inheritance of genomic SV in breeding programs,and support the use of synthetic parents as an important source of novel trait variation.

Phylogeny of Trigonotis in China-with a special reference to its nutlet morphology and plastid genome

The genus Trigonotis comprises nearly 60 species mainly distributed in East and Southeast Asia.China has the largest number of Trigonotis species in the world,with a total of 44 species,of which 38 are endemic.Nutlet morphology is useful for the taxonomic delimitation of Trigonotis.However,there are still controversial circumscriptions of nutlet shape in some species.In previous studies,interspecies phylogenetic relationships were inferred using few DNA markers and very few taxa,which possibly led to erroneous or incomplete conclusions.In this study,the nutlet morphology of 39 Trigonotis taxa and the characteristics of 34 complete chloroplast genomes(29 taxa)were investigated and analyzed.Then,the phylogenetic relationships were discussed within this genus based on complete chloroplast genomes.To the best of our knowledge,this study is the first comprehensive analysis of nutlet morphology and complete chloroplast genome of Trigonotis.Based on nutlet morphology,Trigonotis can be divided into two groups:Group 1,hemispherical or oblique tetrahedron with carpopodiums,and Group 2,inverted tetrahedron without carpopodiums.The chloroplast genome of Trigonotis exhibited a typical quadripartite structure,including 84-86 protein-coding,37 transfer RNA,and 8 ribosomal RNA genes,with a total length of 147,247-148,986 bp.Genes in the junctions were well conserved in Trigonotis,similar to those in other Boraginaceae s.str.species.Furthermore,Trigonotis chloroplast genomes showed relatively high diversity,with more conserved genic regions than intergenic regions;in addition,we detected 14 hot spots(Pi>0.005)in non-coding regions.Phylogenetic analyses based on chloroplast genome data identified highly resolved relationships between Trigonotis species.Specifically,Trigonotis was divided into two clades with strong support:one clade included species with hemispherical or oblique tetrahedron nutlets with carpopodiums and bracts,whereas the other clade included species with inverted tetrahedron nutlets without carpopodiums or bracts.Our results may inform future taxonomic,phylogenetic,and evolutionary studies on Boraginaceae.

Analysis on Genomic Structure Changes and Diversity of Introgression Lines in Dongxiang Wild Rice(Oryza rufipogon Griff.)

[Objective] The aim of the study was to make research on genomic struc- ture variation and variety analysis of Dongxiang wild rice. [Method] Introgression groups of BC1F6 were based on donor of Oryza rufipogon Griff. and receptor of O. sativa sp. indica Kate. Strains of 239 in the group were analyzed on Polymor- phism with the help of 25 couples of SSR primers distributed in 12 pairs of chromo- somes. [Result] Gene fragments of O. rufipogon Griff. were found penetrated in the 25 microsatellite sites and most of the groups kept the parents of Xieqinzao B or DNA sequence of O. rufipogon Griff. The average rate of recurrent homozygous bands was 78.13% in the ILs, but the highest was 94.98% （amplified by primer RM131） and the lowest was 60.25% （RM171）. The average rate of donor homozy- gous bands was 13.37%, but the highest was 32.64% （RM171） and the lowest was 2.93% （RM1095）. There were numerous heterozygous sites in the population and the average heterozygosis rate was 5.62%, while the highest was 10.04%（RM401）. Moreover, we found some parental fragments were lost and some novel fragments were not detected in either parent in BC1F6 population. The average rate of lost bands was 2.88%, while the highest was 13.39% （RM311） and the lowest was 0 （RM401）. The average rate of new bands was 1%. The average of Nei＇s gene di- versity （He） and Shannon＇s Information index （I） were 0.276 and 0.457 respectively in high generation of introgression lines. [Conclusion] The study demonstrated that distant hybridization led to extensive genetic and epigenetic variations in high gener- ation of introgression lines, which expanded the base of genetic variation and laid an important foundation for rice improvement and germplasm innovation.

Phylogenetically clustering of rhizobia by genome structure:application to unclassified Rhizobium

Previous research reveals that the genome structures of rhizobial type strains and reference strains can reflect their phylogenetic relationships. In order to further explore the potential application of genome structure as a phylogenetic marker in rhizobial natural taxonomy, this study analyzed the genome structures of 29 unclassified nodule bacteria isolated from the root nodules of leguminous trees, Robinia sp., Dalbergia spp., and A lbizia spp. and 7 rhizobial reference strains by I-CeuI cleavage, then clustered these bacteria phylogenetically based on their genome structures and compared these clusters with those based on numerical taxonomy and 16S rDNA PCR-RFLP. Eleven phylogenetic clusters were obtained, The clusters were in large part consistent with those based on numerical taxonomy and 16S rDNA PCR-RFLP. Also there are inconsistent clusters based on the above three methods. But results are completely consistent with 16S rRNA clusters. This suggested that the genome structure clustering method can be used to lastly identify root nodule isolates and detect their phylogenetic relationships. The credibility and repeatability of the results, together with the simplicity and possibility to analyze a large number of strains in a short time of the method, indicates the broad potential application of genome structure as phylogenetic marker to categorize rhizobial isolates and should in the future facilitate biodiversity studies.

Genetic Characteristics and Antimicrobial Susceptibility of Arcobacter butzleri Isolates from Raw Chicken Meat and Patients with Diarrhea in China

Arcobacter is an emerging foodborne pathogen worldwide.In this study,the prevalence,antimicrobial susceptibility and genetic characteristics of Arcobacter from different sources were investigated.Eighteen A.butzleri isolates were obtained from 60 raw chicken meat samples(16/60,27%)and 150 patients with diarrhea(2/150,1.3%).The resistance ratios to nalidixic acid,ciprofloxacin,clindamycin,chloramphenicol,and florfenicol were 83.33%(15/18),38.89%(7/18),38.89%(7/18),33.33%(6/18)and 33.33%(6/18),respectively.We performed whole genome sequencing of the 18 isolates,and we predicted antibiotic resistance genes and virulence factors by using assembled genomes through blastx analysis.Two resistance genes,blaOXA-464 and tet(H),and the C254T mutation in gyrA,were identified in the genomes of some resistant isolates.Furthermore,virulence genes,such as flgG,flhA,flhB,fliI,fliP,motA,cadF,cjl349,ciaB,mviN,pldA and tlyA,were found in all strains,whereas hecA,hecB and iroE were found in only some strains.Phylogenetic tree analysis of A.butzleri isolates on the basis of the core-genome single nucleotide polymorphisms showed that two isolates from patients with diarrhea clustered together,separately from the isolates from raw chicken and the chicken strains.This study is the first comprehensive analysis of Arcobacter isolated in Beijing.

Genome-wide evolution of MAPKs family and their expression in response to bacterial infection in seahorse Hippocampus erectus

Seahorses have evolved many unique biological traits,including a male brood pouch,the absence of caudal and pelvic fins,and the lack of spleen and gut-associated lymphatic tissue.The mitogenactivated protein kinases(MAPKs)are known to be involved in various important biological processes including growth,differentiation,immunity,and stress responses.Therefore,we hypothesized that the adaptive evolution and expression of the MAPK gene family in seahorse may differ from those of other teleost species.We identified positive selection sites in the erk2,erk5,jnk1,and p38αMAPK genes of the lined seahorse Hippocampus erectus and tiger-tailed seahorse Hippocampus comes.A novel expression profile of MAPK cascade genes was found in seahorse larvae during the first day after birth based on the RNA-seq data of H.erectus,which refl ected vital signs of immune response to its parental immune system.The expression patterns of the four positively selected MAPK genes were analyzed following the bacterial challenge of Vibrio fortis,revealing their upregulation pattern in brood pouch and other immune tissues.This study enriched our knowledge of the evolution of the H.erectus MAPK subfamilies,and could help better understanding the functional role of MAPKs in teleosts.

The Dicistroviridae: An Emerging Family of Invertebrate Viruses

Dicistroviruses comprise a newly characterized and rapidly expanding family of small RNA viruses of invertebrates. Several features of this virus group have attracted considerable research interest in recent years. In this review I provide an overview of the Dicistroviridae and describe progress made toward the understanding and practical application of dicistroviruses, including (i) construction of the first infectious clone of a dicistrovirus, (ii) use of the baculovirus expression system for production of an infectious dicistrovirus, (iii) the use of Drosophila C virus for analysis of host response to virus infection, and (iv) correlation of the presence of Israeli acute paralysis virus with honey bee colony collapse disorder. The potential use of dicistroviruses for insect pest management is also discussed. The structure, mechanism and practical use of the internal ribosome entry site (IRES) elements has recently been reviewed elsewhere.

Genes encoding heat shock proteins in the endoparasitoid wasp,Cotesia chilonis,and their expression in response to temperatures

Five genes encoding heat shock proteins（HSPs）, Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90, were cloned and sequenced from Cotesia chilonis using RT-PCR and RACE. The c DNA sequences of Cchsp40, Cchsp60, Cchsp70, Cchsc70 and Cchsp90 were 1 265, 2 551, 2 094, 2 297 and 2 635 bp in length, respectively, with a molecular weight（MW） of 39.1, 60.6, 71.45, 70.19 and 82.92 k Da, respectively. The predicted amino acid sequences of these proteins showed high similarities with published HSPs of other insects in Hymenoptera. Analysis of genomic DNAs indicated that Cchsp40, Cchsp60, Cchsp70 and Cchsp90 lacked introns, but Cchsc70 contained an intron. The results also suggested that CcH SP40 in C. chilonis was the Type II HSP40, Cc HSP60 was a member of the mitochondrial HSP60 family, and Cc HSP90 was a part of cytoplasmic HSP90 A family. Expression patterns varied in the five Cchsps in response to temperature. Expression of Cchsp40 and Cchsp60 was induced significantly by cold but not heat stress. Cchsp70 and Cchsc70 showed similar response to the thermal stress and could be induced by both cold and heat, but their expression levels were consistently lower than that of Cchsp40 and Cchsp60. Cchsp90 could be induced by heat stress and mild cold, but not cold stress. In addition, the results demonstrated Cchsc70 might be constitutive and inducible protein that was expressed during normal cell functioning and also up-regulated in response to stressful stimuli while Cchsp70 was solely inducible protein induced by temperature changes. Overall, results generated from this study could significantly advance the understanding of Cchsps in response to temperature and provide important biological information for C. chilonis insects that reared under different temperatures.

Genomic structure analysis of SNC6, a progesterone-receptor associated protein gene, and cloning and characterization of its 5＇-flanking region

Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.

The identification of presence/absence variants associated with the apparent differences of growth period structures between cultivated and wild soybeans

The cultivated soybean（Glycine max（L.） Merr.） was distinguished from its wild progenitor Glycine soja Sieb.＆ Zucc.in growth period structure,by a shorter vegetative phase（V）,a prolonged reproductive phase（R） and hence a larger R/V ratio.However,the genetic basis of the domestication of soybean from wild materials is unclear.Here,a panel of 123 cultivated and 97 wild accessions were genotyped using a set of 24 presence/absence variants（PAVs） while at the same time the materials were phenotyped with respect to flowering and maturity times at two trial sites located at very different latitudes.The major result of this study showed that variation at PAVs is informative for assessing patterns of genetic diversity in Glycine spp.The genotyping was largely consistent with the taxonomic status,although a few accessions were intermediate between the two major clades identified.Allelic diversity was much higher in the wild germplasm than in the cultivated materials.A significant domestication signal was detected at 11 of the PAVs at 0.01 level.In particular,this study has provided information for revealing the genetic basis of photoperiodism which was a prominent feature for the domestication of soybean.A significant marker-trait association with R/V ratio was detected at 14 of the PAVs,but stripping out population structure reduced this to three.These results will provide markers information for further finding of R/V related genes that can help to understand the domestication process and introgress novel genes in wild soybean to broaden the genetic base of modern soybean cultivars.

The first complete organellar genomes of an Antarctic red alga,Pyropia endiviifolia:insights into its genome architecture and phylogenetic position within genus Pyropia(Bangiales,Rhodophyta)

Pyropia species grow in the intertidal zone and are cold-water adapted. To date, most of the information about the whole plastid and mitochondrial genomes(ptDNA and mtDNA) of this genus is limited to Northern Hemisphere species. Here, we report the sequencing of the ptDNA and mtDNA of the Antarctic red alga Pyropia endiviifolia using the Illumina platform. The plastid genome(195 784 bp, 33.28% GC content) contains 210 protein-coding genes, 37 tRNA genes and 6 rRNA genes. The mitochondrial genome(34 603 bp, 30.5% GC content) contains 26 protein-coding genes, 25 tRNA genes and 2 rRNA genes. Our results suggest that the organellar genomes of Py. endiviifolia have a compact organization. Although the collinearity of these genomes is conserved compared with other Pyropia species, the genome sizes show significant differences, mainly because of the different copy numbers of rDNA operons in the pt DNA and group II introns in the mtDNA. The other Pyropia species have 2–3 distinct intronic ORFs in their cox 1 genes, but Py. endiviifolia has no introns in its cox 1 gene. This has led to a smaller mtDNA than in other Pyropia species. The phylogenetic relationships within Pyropia were examined using concatenated gene sets from most of the available organellar genomes with both the maximum likelihood and Bayesian methods. The analysis revealed a sister taxa affiliation between the Antarctic species Py. endiviifolia and the North American species Py. kanakaensis.

Genomic Organization and Transcriptional Regulation of Two Homologous Fucolectin Genes AjFTL-1 and AjFTL-2 in Apostichopus japonicus

F-type lectins(Fucolectins)are carbohydrate-binding proteins and play important roles in innate immune responses against pathogenic microbial invasion.In our previous research,we found that two homologous Fucolectin genes,AjFTL-1 and AjFTL-2,exhibited different expression profiles after lipopolysaccharides(LPS)challenge in Apostichopus japonicus.However,the transcriptional regulation mechanism of these two genes remains largely unknown.In this study,the 5'flanking regions of Aj FTL-1 and Aj FTL-2 genes were cloned and the promoter activities were studied in epithelioma papulosum cyprinid(EPC)cell system.First,in silico analysis indicated that these two promoters both contain numerous putative transcription factor binding sites including NF-κB,CREB,and CREBP1,and both contain a TATA box.Additionally,luciferase assay and progressive 5'truncation analysis revealed that AjFTL-1 and AjFTL-2 both possess high promoter activities in EPC cells.Moreover,the luciferase activity of AjFTL-1 promoter was significantly regulated by peptidoglycan(PGN)and mannan(MAN),while AjFTL-2 promoter was prominently regulated by LPS and MAN,indicating Aj FTL-1 and Aj FTL-2 genes showed different transcriptional regulation pattern under different immune stimulation.More importantly,analyses of the functional promoter regions revealed the presence of two potential NF-κB binding sites(-769 bp to-761 bp,-185 bp to-172 bp)in Aj FTL-1 and one potential binding site(-530 to-517 bp)in Aj FTL-2.Different truncated reporter vectors and expression vector co-transfection revealed that transcription factor NF-κB/Rel could significantly increase the AjFTL-2 promoter activity,but not AjFTL-1 promoter activity.These findings indicated that in marine invertebrates,different Fucolectin members differ in transcription regulations and expression patterns,and might play different roles in immune defenses during pathogen infection.

Relationship between Genomic Types of Escherichia coli and Clinical Diseases

In this study, by analysis of genome structures of E. coli, the relationships between the genomic types of E. coli and the associated diseases were investigated. Samples of sputum, urine and other excretions from patients with different infective diseases were collected. And 62 E. coli strains were isolated from these samples. Intact bacterial genomic DNA was cleaved with I-CeuI, separated by pulsed field gel electrophoresis and then typed on the basis of cleavage map. The results showed that 7 I-CeuI sites were found in all the genome structures of the 62 E. coli, indicating that there were 7 rrn operons in the genomes. The size of genome ranged from 4500 kb to 5000 kb. According to the genome structures, 62 E. coli strains were divided into 30 genome types. It was concluded that genome structures of E. coli isolated from the patients with different infective diseases varied to some extent, suggesting that some genome types of E. coli were closely related to some infective diseases.

Intelligent Approach for Clustering Mutations’ Nature of COVID-19 Genome

In December 2019,a group of people in Wuhan city of Hubei province of China were found to be affected by an infection called dark etiology pneumonia.The outbreak of this pneumonia infection was declared a deadly disease by the China Center for Disease Control and Prevention on January 9,2020,named Novel Coronavirus 2019(nCoV-2019).This nCoV-2019 is now known as COVID-19.There is a big list of infections of this coronavirus which is present in the form of a big family.This virus can cause several diseases that usually develop with a serious problem.According to the World Health Organization(WHO),2019-nCoV has been placed as the modern generation of Severe Acute Respiratory Syndrome(SARS)and Middle East Respiratory Syndrome(MERS)coronaviruses,so COVID-19 can repeatedly change its internal genome structure to extend its existence.Understanding and accurately predicting the mutational properties of the genome structure of COVID-19 can form a good leadership role in preventing and fighting against coronavirus.In this research paper,an analytical approach has been presented which is based on the k-means cluster technique of machine learning to find the clusters over the mutational properties of the COVID-19 viruses’complete genome.This method would be able to act as a promising tool to monitor and track pathogenic infections in their stable and local genetics/hereditary varieties.This paper identifies five main clusters of mutations with k=5 as best in most cases in the coronavirus that could help scientists and researchers develop disease control vaccines for the transformation of coronaviruses.

Toward Elucidating the Structure of Tetraploid Cotton Genome

Upland cotton has the highest yield,and accounts for >95% of world cotton production.Decoding upland cotton genomes will undoubtedly provide the ultimate reference and resource for structural,functional,and evolutionary studies of the species.Here,we employed GeneTrek and

Twenty years of rice genomics research: From sequencing and functional genomics to quantitative genomics

Since the completion of the rice genome sequencing project in 2005,we have entered the era of rice genomics,which is still in its ascendancy.Rice genomics studies can be classified into three stages:structural genomics,functional genomics,and quantitative genomics.Structural genomics refers primarily to genome sequencing for the construction of a complete map of rice genome sequence.This is fundamental for rice genetics and molecular biology research.Functional genomics aims to decode the functions of rice genes.Quantitative genomics is large-scale sequence-and statistics-based research to define the quantitative traits and genetic features of rice populations.Rice genomics has been a transformative influence on rice biological research and contributes significantly to rice breeding,making rice a good model plant for studying crop sciences.

Impact of Genomics and in Silico Related Technologies in the Drug Discovery Process

In order to evaluate to what extent will genomics and in silico related technologies improve overall drug discovery process, we analyzed three studies comparing cost, time and attrition rate at each step of the drug discovery process, between standard pharmaceutical and genomics based approaches.

Snapshot of Structural Variations in the Tibetan Wild Boar Genome at Single-Nucleotide Resolution

Genomic structural variations （SVs）, particularly insertions, deletions and inversions, can contribute to the heterogeneity of millions of nucleotides within a genome, and are likely to make an important contribution to biological diversity and phenotypic variation （Alkan et al., 2011; Bickhart and Liu, 2014）. With the rapid development of the next-generation sequencing technologies and the new assembly methodolo- gies, the multiple de novo assemblies of genomes within a species allow researchers to explore more detailed SV maps （Li et al., 2011）. Compared with the traditional read depth algorithm using the whole-genome resequencing approach and array-based technologies （Baker, 2012; Wang et al., 2012;

Structural and Expressional Variation Analyses of Mitochondrial Genomes Reveal Candidate Transcripts for the S^V Cytoplasmic Male Sterility in Wheat(Triticum aestivum L.)

Common wheat （Triticum aestivum L.） is one of the most important crops, and intra-specific wheat hybrids have obvious heterosis in yield and protein quality. Therefore, utilization of hybrid wheat varieties offers an effective way to increase yield and nutrition. Cytoplasmic male sterility （CMS） systems are a useful genetic tool for hybrid crop breeding, and are ideal models for studying the genetic interaction and cooperative function of mitochondrial and nuclear genomes in plants （Schnable and Wise, 1998; Hanson and Bentolila, 2004）.

3D genome structural variations play important roles in regulating seed oil content of Brassica napus

Dissecting the complex regulatory mechanism of seed oil content(SOC)is one of the main research goals in Brassica napus.Increasing evidence suggests that genome architecture is linked to multiple biological functions.However,the effect of genome architecture on SOC regulation remains unclear.Here,we used high-throughput chromatin conformation capture to characterize differences in the three-dimen-sional(3D)landscape of genome architecture of seeds from two B.napus lines,N53-2(with high SOC)and Ken-C8(with low SOC).Bioinformatics analysis demonstrated that differentially accessible regions and differentially expressed genes between N53-2 and Ken-C8 were preferentially enriched in regions with quantitative trait loci(QTLs)/associated genomic regions(AGRs)for SOC.A multi-omics analysis demonstrated that expression of SOC-related genes was tightly correlated with genome structural varia-tions in QTLs/AGRs of B.napus.The candidate gene BnaA09g48250D,which showed structural variation in a QTL/AGR on chrA09,was identified byfine-mapping of a KN double-haploid population derived from hybridization of N53-2 and Ken-C8.Overexpression and knockout of BnaA09g48250D led to significant in-creases and decreases in SOC,respectively,in the transgenic lines.Taken together,our results reveal the 3D genome architecture of B.napus seeds and the roles of genome structural variations in SOC regulation,enriching our understanding of the molecular mechanisms of SOC regulation from the perspective of spatial chromatin structure.