Leishmania and other trypanosomatids, the mtDNA  is composed of thousands of minicircles and a few maxicircles, catenated together into a complex network. Maxicircles are functionally similar to other eukaryotic mtDNAs, whereas minicircles are involved in RNA editing of some maxicircle-encoded transcripts. Next-generation sequencing (NGS) is increasingly used for assembling nuclear genomes and, currently, a large number of genomic sequences are available. However, most of the time, the mitochondrial genome is ignored in the genome assembly processes. The aim of this study was to develop a pipeline to assemble Leishmania minicircles and maxicircle DNA molecules, exploiting the raw data generated in the NGS projects. As a result, the maxicircle molecules and the plethora of minicircle classes for Leishmania major, Leishmania infantum and Leishmania braziliensis have been characterized. We have observed that whereas the heterogeneity of minicircle sequences existing in a single cell hampers their use for Leishmania typing and classification, maxicircles emerge as an extremely robust genetic marker for taxonomic studies within the clade of kinetoplastids.The mitochondrial DNA (mtDNA), which is present in almost all eukaryotic organisms, is a useful marker for phylogenetic studies due to its relative high conservation and its inheritance manner. In  Leishmania, classified within the order Trypanosomatida, belongs to the lineage Excavata, one of the earliest branches diverging from the eukaryotic tree [Trypanosoma ssp., causative agents of Chagas disease and sleeping sickness in humans, and Leishmania spp., which cause several types of leishmaniasis [The genus tic tree . The trytic tree , includimaniasis . Anothermaniasis . DNA maxmaniasis . The gRNmaniasis ,6. Nevermaniasis ,8.In order to completely edit the maxicircle transcripts, a large number of different minicircles are needed for providing the complete set of gRNAs. If one class of minicircles is lost, the parasite cannot complete the editing of the corresponding maxicircle transcripts and therefore it may die. To minimize fatal losses, each class of minicircles is present in several copies and redundant gRNAs are also observed . MoreoveLeishmania tarentolae has been widely used as a model organism for studying RNA editing, and, as a consequence, the structure of its mitochondrial genome (maxicircle and minicircles) is the best characterized to date among the trypanosomatids. Approximately, there are 5000–10000 minicircles and 20–50 maxicircles within the sole mitochondrion existing in this parasite [L. tarentolae, was sequenced several years ago [L. tarentolae maxicircle [L. tarentolae maxicircle (around 12 kb) consists of tandem repeats of varying complexities [ears ago . The idexicircle , and thexicircle . Five ofxicircle . A largelexities . This relexities .L. tarentolae, a parasite for the lizard Tarentola mauritanica, the coding region of the maxicircle has not been completely sequenced for other Leishmania species until very recently (see below). Regarding maxicircles from human pathogenic Leishmania species, in 2008, the sequence of an 8.4 kb fragment, belonging to the coding region of the Leishmania major maxicircle, was reported [L. donovani (LdBob strain) maxicircle was determined; a notable particularity found in the sequenced maxicircle was a full-size minicircle integrated in the ND1 gene [Leishmania mexicana amazonensis (LV78 strain), though the complete set of mitochondrial pan-edited mRNAs has been characterized, the maxicircle sequence assembly has not been reported [Leishmania braziliensis maxicircle were assembled [L. braziliensis regarding other pathogenic Leishmania species [Leishmania species and strains [Leishmania species named to date. In this work, we describe a pipeline designed to assemble the complete maxicircle sequences and the different minicircles by using the genomic NGS-reads generated during Leishmania nuclear genome sequencing. For this objective, three of the more pathogenic Leishmania species were selected: Leishmania infantum, causative agent of visceral leishmaniasis in the Mediterranean Basin, the Middle East, central Asia, South America and Central America, L. braziliensis, which causes metastasizing mucocutaneous leishmaniasis in South America, and L. major, responsible for cutaneous leishmaniasis in Iran, Saudi Arabia, North Africa, the Middle East, Central Asia and West Africa [L. major (Friedlin strain), L. infantum (JPCM5 strain) and L. braziliensis (M2904 strain), and many of the distinct minicircles existing in these species. Finally, we assessed the usefulness of both types of mitochondrial sequences for phylogenetic analyses. A robust phylogenetic tree was constructed based on the complete coding-region from the relatively few maxicircles to date sequenced and characterized in kinetoplastids.Apart from reported . Soon afND1 gene . For Leireported . Two fra species , a remar strains ,30,31,32t Africa . As a reL. major (MHOM/IL/81/Friedlin), L. infantum  and L. braziliensis (MHOM/BR/75/M2904) were provided by Dr. J. Moreno . Promastigotes were cultured at 26 °C in M199 medium (Sigma-Aldrich), supplemented with 10% heat-inactivated fetal calf serum. Genomic DNA was isolated following the classical phenol-chloroform-isoamyl alcohol extraction method as described elsewhere [Promastigote cultures of lsewhere .Leishmania species were: 53,340,123 for L. major; 56,833,213 for L. infantum and 51,050,312 for L. braziliensis. Further information about the nuclear genome sequencing and assembly for these three species is available at http://leish-esp.cbm.uam.es/ and in previous publications [Construction of libraries from total DNA and paired-end library sequencing were performed at the Centro Nacional de Análisis Genómico  using Illumina HiSeq 2000 technology. Reads having unexpected combinations of indices (due to barcode swapping or index hopping) were removed from the data. The number of 2 × 125 nucleotide (nt) reads generated for each ications ,32.Leishmania genome sequences available at Leish-ESP website (http://leish-esp.cbm.uam.es), TriTrypDB (https://tritrypdb.org), NCBI (https://www.ncbi.nlm.nih.gov) and EMBL-EBI (https://www.ebi.ac.uk/). The SAM files were converted into BAM files using Samtools (http://www.htslib.org/) and the unaligned reads were extracted using bedtools2 [http://prinseq.sourceforge.net/) was applied to quality filtering/trimming of the unaligned reads , and only reads with length ≥60 nt were used. The exact parameters were: -trim_qual_right 25, -trim_qual_left 25, -trim_qual_type mean, -trim_qual_window 5, -trim_qual_step 1, -trim_ns_left 1, -trim_ns_right 1, -ns_max_p 1, -ns_max_n 3, -min_qual_mean 25 and -min_len 60. Pair reads were selected and ordered using in-house scripts. These reads were used to generate assemblies (contigs) by the IDBA_UD (version 1.1.1) assembler [Illumina reads were aligned by Bowtie2  against edtools2 . Prinseqssembler , selectiPutative minicircle contigs were further tested for circularity, manifested by the presence of direct repeated ends in the assembled molecule. Briefly, contigs were split into two halves, and then Minimus2 (from toAmos (version 3.1.0)) tool was used to identify repeated ends as described elsewhere . Finallyhttps://github.com/genomics-ngsCBMSO/Mithocondrial-assembly.git).The complete pipeline, as well as the associated in-house scripts, have been deposited in GitHub  were designed within the conserved region of LmjF-mic1; both oligonucleotides overlap each other by 4 nt at the 5′-end. Amplification was carried out in a 50 µL-final volume using the Phusion Hot-Start II DNA polymerase (ThermoFisher Scientific) and 100 ng of total DNA; PCR was run on a Techne TC-3000 PCR Thermal Cycler using the following profile: Initial denaturation at 98 °C for 30 s followed by 35 cycles of 10 s at 98 °C, 30 s at 66.5 °C and 45 s at 72 °C, completed by a final incubation of 5 min at 72 °C. The amplification products were purified using the Favorgen FavorPrep GEL/PCR Purification kit and cloned into pNZY28 vector using the NZY-blunt PCR cloning kit (Nzytech). After transformation of Escherichia coli DH5α bacteria and antibiotic selection, plasmid DNA from bacteria cultures was extracted using the AccuPrep Plasmid MiniExtraction kit . Clones were checked by restriction endonuclease analysis and sequenced at the Genomic Unit from Universidad Complutense de Madrid. The clone sequences were edited using Bioedit Software  and then compared to the in-silico assembled sequences.Experimental validation of the precise overlapping between the two contigs (LmjF_max-CgL and LmjF_max-CgS) containing https://www.ebi.ac.uk/Tools/msa/clustalo/). Manual refinements were omitted. Phylogenetic relationships were inferred by using the maximum likelihood method and Tamura-Nei model [Phylogenetic trees were constructed using either the full sequence of minicircles or the entire coding region of the maxicircles. The sequences were aligned using Clustal Omega  tool [The conserved regions of minicircles for each one of the species, .4) tool . For theL. braziliensis maxicircle, LR697136 for the L. adleri maxicircle, LR697135 for L. guyanensis maxicircle, LR697137 for L. infantum maxicircle and LR697138 for the L. major maxicircle. Additionally, the sequences for the L. major, L. infantum and L. braziliensis maxicircles and minicircles can be downloaded at the Website Leish-ESP (http://leish-esp.cbm.uam.es).The sequencing NGS-reads (used for the assemblies reported in this work) and the assembled sequences have been submitted to the European Nucleotide Archive (ENA) and are available under the study accession number PRJEB33887. The ENA/GenBank accession numbers of the assembled sequences are: LR697134 for the Leishmania whole-DNA sequencing experiment. For this purpose, we used the reads derived from an on-going project aimed to the de novo assembly of the L. major (Friedlin strain) nuclear genome (manuscript in preparation). Based on a strategy previously used for uncovering seven misassembled regions in the L. major nuclear genome using RNA-seq reads [L. major whole-DNA sequencing, were mapped to the reference genome . Above four million failed to align and, after further filtering by quality, 3,793,390 pair-end reads were selected to carry out a de novo assembly. As a result, 153 contigs longer than 500 nt were obtained. Two of them showed sequence identity with maxicircle entries found in the GenBank/ENA databases. The longest one, named LmjF_max-CgL (17273 nt in length) was found to include the 8738 nt maxicircle sequence described by Yatawara et al [Trypanosoma brucei EATRO strain 427 the maxicircle sequence has been completed, yielding a molecule of 23016 bp in length [L. major (Friedlin) maxicircle would have a size around 31 kb in length.The initial question of this work was whether the assembly of the mitochondrial genome  would be possible using the sequence reads generated by the Illumina platform in a eq reads , we desira et al ,44. To tn length . AccordiL. tarentolae maxicircle (GenBank accession number M10126.1) showed that the coding region of the assembled L. major maxicircle was highly conserved in both sequence and gene order. L. major maxicircle. L. major assembled sequence and the reads coverage. The depth-coverage of reads on the region located upstream of the coding region in the maxicircle contig evidenced the low complexity and repetitive nature of the DR region.BLAST alignments against the L. major maxicircle prompted us to repeat the strategy for two other Leishmania species, L. infantum and L. braziliensis, whose genome assemblies have been recently improved by our group [Leishmania species . After mapping and filtering, 4,057,401 and 1,347,958 paired-end reads for L. infantum and L. braziliensis, respectively, remained as non-aligned reads. These reads were used for the de novo assembly into contigs. For each species, a large contig and several small ones were generated. The large contigs were found to contain all the maxicircle genes . According to an author’s comment accompanying the entries, these sequences were assembled from Illumina reads using the Velvet tool. Interestingly, but not surprisingly as both studies have been done using the same strain, sequence identities higher than 99% exist between LbrM-mic1 and KY698625 sequence, LbrM-mic3 and KY698802, and LbrM-mic5 and KY698812.In recent work, Kocher and co-workers  carried Leishmania species. Although the size range in the L. major minicircles was higher than those observed for the L. infantum and L. braziliensis minicircles, the mean size was clearly shorter in L. major, 691 nt versus 797 nt and 744 nt for the L. infantum and L. braziliensis minicircles, respectively. Furthermore, the values calculated from the 42 L. braziliensis (M2904 strain) minicircles deposited in the GenBank database (see above) were similar (size range of 738 to 763 nt and a mean size of 748 nt) to those assembled in this work.Leishmania species. For this study, the 42 sequences for L. braziliensis minicircles available at GenBank (see above) were used. The results, shown in L. braziliensis minicircles, a second CSB-2-like motif (CCCCGTGC) was found to be well conserved , a PCR-based experiment was conducted to validate the correctness of the rcle see A, it wasrcle see B, four rrentolae .L. major  method was conducted. As shown in Leishmania typing based on sequencing a single minicircle may lead to miss-identification, considering that several distinct minicircle classes coexist in a single parasite.Firstly, a phylogenetic tree was generated from the 97 complete minicircles assembled for L. major . The picLeishmania and related trypanosomatids might be consistent and reliable. To investigate this possibility further, we inspected GenBank and retrieved those maxicircle sequences that contain the entire gene-coding region. However, among the large number of maxicircle sequences currently deposited in this database, only thirteen were found to cover the gene-coding region completely. These sequences, together with the five maxicircles assembled in this work, were used to construct a phylogenetic tree using the ML method  from NGS reads generated by sequencing of total DNA


Vigna represented by more than 100 species is a source of nutritious edible seeds and sprouts that are rich sources of protein and dietary supplements. It is further valuable because of therapeutic attributes due to its antioxidant and anti-diabetic properties. A highly diverse and an extremely ecological niche of different species can be valuable genomic resources for productivity enhancement. It is one of the most underutilized crops for food security and animal feeds. In spite of huge species diversity, only three species of Vigna have been sequenced; thus, there is a need for molecular markers for the remaining species. Computational approach of microsatellite marker discovery along with evaluation of polymorphism utilizing available genomic data of different genotypes can be a quick and an economical approach for genomic resource development. Cross-species transferability by e-PCR over available genomes can further prioritize the potential SSR markers, which could be used for genetic diversity and population differentiation of the remaining species saving cost and time. We present VigSatDB—the world’s first comprehensive microsatellite database of genus Vigna, containing >875 K putative microsatellite markers with 772 354 simple and 103 865 compound markers mined from six genome assemblies of three Vigna species, namely, Vigna radiata (Mung bean), Vigna angularis (Adzuki bean) and Vigna unguiculata (Cowpea). It also contains 1976 validated published markers. Markers can be selected on the basis of chromosomes/location specificity, and primers can be generated using Primer3core tool integrated at backend. Efficacy of VigSatDB for microsatellite loci genotyping has been evaluated by 15 markers over a panel of 10 diverse genotype of V. radiata. Our web genomic resources can be used in diversity analysis, population and varietal differentiation, discovery of quantitative trait loci/genes, marker-assisted varietal improvement in endeavor of Vigna crop productivity and management.Genus  Vigna is one of the most important Fabaceae family crops, which is mostly found in tropical regions and warm temperate region of Asia and Africa , cowpea (Vigna unguiculata), adzuki bean (Vigna angularis) and urdbean (Vigna mungo) are the major domesticated species. The vast genetic diversity in wild species of Vigna is its best attractive feature, and these wild species are highly tolerant to biotic and abiotic stress such as pests, diseases, drought, alkaline soil and high salinity. Since more than 90 species of this genus are well adaptive to very extreme ecological niches like marine beach, deserts and swamps, they could be a potential source of genomic resource for productivity enhancement , adzuki bean (V. angularis var. angularis) and V. unguiculata (Cowpea) were sequenced. In genus Vigna, mostly are diploid (2n = 2x = 22), while in Vigna reflexo-pilosa, mostly are tetraploid (2n = 4x = 44). The genome size of all the Vigna species may vary from species to species ranging from 416 to 1394 Mb , also known as microsatellites, are the co-dominant markers and plays an important role in biological functions. It is present in the stretch of DNA sequences that contain 1–6 repeating units of nucleotides . These rVigna  and linkage mapping  agreement and intellectual property rights, plant breeders have their ownership of variety on the basis of distinctiveness, uniformity and stability (DUS) features, which can be used for assigning of new variety status as well as resolving legal varietal disputes . MicrosaSince flanking region of microsatellite markers are well conserved across closely related species, heterologous PCR primers of such locus can cater the immediate need of molecular markers most economically . MoleculVigna species like mungbean  and V. angularis (cv. Jingnong 6), it has several limitations like lack of automation for SSR mining, designing of primers, input query sequence limited to 30 Kb  contains 110 plant species that includes two Vigna, namely, V. radiata (mung bean), V. angularis (adzuki bean) and V. unguiculata (cowpea). It further aims at development of a web genomic resource having option for chromosome-wise SSR mining and primer designing for genotyping along with e-PCR-based polymorphism discovery. It also aims to provide annotated genic region SSR to be used as functional domain markers (FDMs).Present work aims at genome-wide mining of SSR markers in three species of genus V. radiata (mung bean), V. angularis (adzuki bean) and V. unguiculata (cowpea) were retrieved from NCBI   , whereasIT213134 ,30, 31.cultivar . Out of SSRs were mined using perl script of MISA (MIcroSAtellite identification tool) with default parameters such as 10 repeating units for mono, 6 repeating units for di and 5 repeating units for tri, tetra, penta and hexa . MarkersVigna, namely V. radiata (cv. VC1973A) and V. angularis (cv. Jingnong 6) that are fully annotated for gene function were incorporated in genome browser in the database. This uses general feature format coordinates of each gene file over coordinates of mined SSRs and displays gene ID along with structural and functional details.Available two species of in silico polymorphic SSR within the two species, namely V. radiata (VC1973A and RIL59) and V. angularis . Cross-species transferability was evaluated over genomic DNA sequence of V. angularis and V. unguiculata by e-PCR of microsatellite loci obtained from V. radiata cv. VC1973A. For this evaluation, loci were selected based on the following criteria: simple di-nucleotide repeat as they are expected to have more polymorphism due to higher probability of slippage in DNA replication . This search is based on two modules for primer selection, namely, `self-designed primers’ and `External primers’. The `self-designed primers’ search generates new primers. VigSatDb has the references genome of six assembles with user-friendly option for uploading of genotype file for polymorphic discovery. Both, forward and reverse primers along with marker are located in the genotype file. The markers with different product size in the reference as well as genotype file are considered to be polymorphic markers. The second module, `External primers’ is based on primers that are already published. An in-house perl script has been written to detect the polymorphic markers by searching existing primers of loci in the flanking region of reference genome file. Then the e-PCR product sizes are computed and displayed as polymorphic loci. Using R code, provision of simulated gel has been made. Moreover, all the results can be achieved as HTML files, downloaded directly or through mail.Vigna microsatellite database (VigSatDB) a `three tier architecture’ relational database has been developed having client tier, middle tier and database tier. Data of predicted markers and experimentally validated published markers were stored in the backend in MYSQL database and launched on apache server. The web server contains seven tabs viz. Home, Microsatellite, Tools, Polymorphism, Statistics, Tutorial and Team. The database is further linked to web interface using open source PHP scripting language. Primer3core executable was incorporated into the database for primer generation. It gives the output having three sets of primers with their respective PCR product size, GC content, melting point and start position , namely NMK1512, SKM 1508, GSM1715, VIRAT, GAM5, GM-4, K-857, GJM1712, MEHA and VMS-15-1. These varieties were collected from Main Pulse Research Station, Junagadh Agricultural University, Junagadh, Gujarat, India. Young leaves were used to isolate DNA by CTAB method using protocol as described by Doyle and Doyle  along with 1 μl (1 μM) each of SSR forward and reverse primers. SSR-PCR product was further analyzed on a capillary electrophoresis  using QIAxcel DNA Screening Kit and the images were analyzed using QIAxcel ScreenGel Software.PCR reaction for all the microsatellite loci were carried out in a 20-μl PCR reaction mixture containing 4 μl 2.5 mM dNTPs, 1 μl of 20 ng DNA in 2.5 μl 10× Taq polymerase assay buffer, 1.5 μl MgClSSR loci were mined successfully using MISA. SSR loci details were also mined from the public domain literature. Web genomic resource was developed with provision to generate primers for genotyping of loci. A total of 876 219 putative microsatellite markers were mined having 772 354 simple and 103 865 compound repeats . Genotypviz., V. radiata (VC1973A), V. angularis (Jingnong 6) and V. angularis (Kyungwonpat), was used for the initial mining of SSR loci. Species and genotype-wise variation in abundance of SSR loci are shown in The genotype having chromosome-wise whole-genome data of all three species, In cv. VC1973A, maximum repeats were found on chromosome 7 (26 280), followed by chromosome 8 (20 521) and chromosome 5 (18 212). Chromosome 3 had minimum number of repeats (6579) having maximum SSR density per Megabase (Mb) (573.2). The least SSR density was found on chromosome 6 (411.3).Although Jingnong 6 and Kyungwonpat cultivars were from the same species, at chromosome level substantial variation in SSR abundance was observed. In Jingnong 6, maximum markers were found on chromosome 1 (14 964), while minimum markers were observed on chromosome 6 (7391). In the case of cv. Kyungwonpat, chromosomes 6 and 10 were found to be with maximum (13 887) and minimum (3102) markers, respectively . In the V. radiata. Similarly, in species V. angularis, the two sets of comparison between genotypes Jingnong 6 and Kyungwonpat and Jingnong 6 and IT213134 revealed 243 and 195 polymorphic loci, respectively  than V. unguiculata (2373). In order to obtain more SSR loci having cross-species transferability, the second species (V. angularis) was treated as focal species against non-focal species (V. unguiculata), which revealed 5882 loci  and hexa-nucleotides were the lowest (177) in number. In V. angularis cv. Jingnong6, maximum numbers of SSRs were mono-type (86 204), while minimum number of SSR markers belonged to hexa-type (247). In V. unguiculata, mono-nucleotides were highest (59 935), while hexa-nucleotides were lowest (122) in number. In all the three species, mono-nucleotide repeats were most abundant followed by di- and tri-nucleotide repeats  and mashbean (V. mungo)  . SSR has. mungo) .V. mungo) has been used to differentiate more than 30 varieties with high precision by rapid genotyping in DUS testing program of germplasm management (V. radiata) (V. radiata)  . Using fradiata) . Putativugarcane , sesame ugarcane , capsicuugarcane , tea 57V. mungo ugarcane  and eggpugarcane .Vigna marina, SSR has been used to discover one major QTL controlling salt tolerance, and such QTL can be transferred in breeding program using flanking region SSR markers having allele size difference between donor and recipient varieties  such conservation is reported in tune of more than one-fifth. Cross-species transferability of SSR loci in genus Vigna (V. angularis) is reported in tune of more than two-thirds in species like V. umbellata, V. mungo, V. aconitifolia and V. radiata (V. aconitifolia and V. reflexo-pilosa (Vigna is due to their higher homology at genomic level; thus, they are much more useful in comparative genomics and genome mapping (Vigna.Although there are several known limitations in the utilization of cross-species SSR or heterologous SSR in diversity analysis , it is i radiata . This exo-pilosa . Higher  mapping . Very saVigna covering three of its commercially important species. VigSatDB contains a total of 876 219 putative microsatellite DNA markers, which also includes 1976 validated published markers. A total of 10 457 polymorphic markers obtained by e-PCR across genotypes can be used for germplasm differentiation and diversity analysis saving in vitro cost and time required for in vitro polymorphic discovery. Our finding on cross species transferability of microsatellite locus among three different species of Vigna can be used to cater the need of molecular markers especially for more than 100 species of genus Vigna where there is no whole-genome sequence data even today. This genomic resource can be of immense use for the global community. It can be used for chromosome-wise microsatellite locus mining and primer designing for non-genic and genic FDM-SSR for rapid genotyping. It can also be used to accelerate polymorphism discovery by e-PCR most economically especially needed in future re-sequencing projects. It can be used not only for knowledge discovery research like QTL and gene mapping but also for marker-assisted breeding in Vigna germplasm improvement and management.We report here first comprehensive web genomic resource of genus in vitro validation of markers. R.S.J., M.A.I. and S.J. drafted the manuscript. D.K., S.J., P.K.Y., M.A.I. and A.R. edited the manuscript. All authors read and approved the final manuscript.P.K.Y., D.K., M.A.I., A.R., S.J. and R.S.J. conceived the theme of the study. R.S.J., M.A.I. and S.J. did the computational analysis of generated data. R.S.J., U.B.A. and V.A. contributed to database development. S.B.B. and R.S.T. did Supplementary_Table_1_baz055Click here for additional data file.Supplementary_Table_2_baz055Click here for additional data file.supplementary_Table_3_baz55Click here for additional data file.


Castanea mollissima Blume) can be infested by Dryocosmus kuriphilus Yasumatsu, resulting in gall formation and yield losses. Research on the control of gall wasps using genomics approaches is rarely reported. We used RNA-seq to investigate the dynamic changes in the genes of a chestnut species (C. mollissima B.) during four gall-formation stages caused by D. kuriphilus. A total of 21,306 genes were annotated by BLAST in databases. Transcriptome comparison between different gall-formation stages revealed many genes that were differentially expressed compared to the control. Among these, 2410, 7373, 6294, and 9412 genes were differentially expressed in four gall-formation stages: initiation stage (A), early growth stage (B), late growth stage (C), and maturation stage (D), respectively. Annotation analysis indicated that many metabolic processes  were affected. Interesting genes encoding putative components of signal transduction, stress response, and transcription factors were also differentially regulated. These genes might play important roles in response to D. kuriphilus gall formation. These new data on the mechanism by which D. kuriphilus infests chestnuts could help improve chestnut resistance.Chinese chestnut ( Castanea mollissima), Japanese chestnut (Castanea crenata), European chestnut (Castanea sativa), and American chestnut (Castanea dentata) [Castanea mollissima BL) is a major cultivated species producing edible chestnuts in China [C. mollissima has been affected by major diseases and pests which have caused significant losses. The chestnut gall wasp, Dryocosmus kuriphilus (Hymenoptera Cynipidae), which is native to mainland Asia, was introduced to Japan, North America, and Europe and has become a significant pest of cultivated chestnuts [D. kuriphilus can prevent normal plant growth and cause a progressive loss of photosynthetic biomass [Chestnuts are economically important nuts and timber-producing trees that include the Chinese chestnut (dentata) ,2. Chinein China . Howeverhestnuts . D. kuri biomass . Hence, Insect galls are composed of plant tissues that develop in response to insect-derived stimuli, and present inducer-specific phenotypes and patterns of tissue differentiation . Gall deCastanea mollssina Blume) and D. kuriphilus still need further study.In the battle between plants and agents, plants have evolved immune systems that can recognize potentially intrusive agents and initiate effective defense responses . Host chGenomic tools, such as transcriptome analysis, can aid in the identification of resistance genes and the development of blight-resistant chestnut . High-thD. kuriphilus. The gall-formation stages mentioned above are easy to differentiate visually and are natural sampling points for investigating the interaction between pest and host plant tissues during gall development. However, in this study, considering the gall grows rapidly during the growth stage, we hypothesize that the gene expression of the plant host will also change dramatically during the growth stage. Hence, we divided the growth stage into early growth stage and late growth stage visually. Finally, we focused on four gall-formation stages: initiation (A), early growth (B), late growth (C), and maturation (D) stage to investigate the dynamic changes of gene expression of the Chinese chestnut (Castanea mollssina Blume). Differentially expressed genes (DEGs) analysis enabled identification of a large number of candidate pathogen-response genes in Chinese chestnut for use in studying pathways involved in resistance to D. kuriphilus. This host–insect interaction may be important in breeding programs for resistant genotypes. Also, information about host responses is useful to advance our understanding of the mechanism by which D. kuriphilus affects other species of trees.Hence, in this study, we investigated changes in the Chinese chestnut gall transcriptome induced by D. kuriphilus are shown in Phenotype changes of the different development stages of gall formation by To obtain a global overview of the Chinese chestnut transcriptome during leaf gall formation, a total of 15 cDNA libraries were constructed and sequenced on a Illumina Hiseq 2500 platform. After removing adaptors and low-quality sequences, a total of 295.14 million clean reads were obtained. On average, 23.94 million clean reads were obtained from each sample . The perTo test sample repeatability, we calculated the correlation coefficient between the samples. The correlation coefficient in the repeat group reached 0.99 or higher , indicatUsing the criteria of Fold change ≥2 and divergence probability ≥0.8, a total of 2410, 7373, 6294, and 9412 genes were differentially expressed in the four gall-formation stages compared to the control . The fouThe Venn diagram  shows thTo further explore the trends of these differentially expressed genes, we used the R package pheatmap to draw heat maps of DEGs a. DEGs wAll of the DEGs were BLAST against the KEGG Ontology (KO) database. At stage A, 1035 annotated DEGs were classified into 130 biological pathways belonging to 20 KEGG categories a. At stap < 0.05, FDR < 0.05). Enrichment of “plant–pathogen interaction”, “Plant hormone signal transduction”, and “phenylpropanoid biosynthesis” was found with a threshold of false discovery rate (FDR) < 0.05  multiple test correction to analyze KEGG enrichment for all of the DEGs in four stages  < 0.05 . Furtherp < 0.05 .C. mollissima. In JA signaling, there were four genes encoding a transcription factor that were differentially expressed at stage A, with three upregulated and one downregulated  metabolism-related genes showed different expression patterns during gall formation. Four  stage D . In contormation . Another stage D . Some we stage D . Besideshestnuts .GDSL esterase/lipase, and most of them were downregulated during the gall formation process  to mature stage (stage D), and two were slightly upregulated only at stage C  genes were also upregulated, with only one downregulation. One cinnamyl-alcohol dehydrogenase (CAD) and two shikimate O-hydroxycinnamoyltransferase were identified with upregulation during the whole gall formation process. Four caffeic acid O-methyltransferase (COMT) were identified, with three upregulated and only one downregulated.Transcripts responsible for phenylpropanoid biosynthesis were active during gall formation on the plant host. All the  process . Four caWRKY1, bHLH61, and pathogenesis-related protein1) showed different expression patterns between these two approaches at the first two gall stages. RNA-seq analysis was mainly used for high-throughput screening and indicated the global gene expression patterns of different samples. Hence, the mismatch of the expression profile shown by the RNA-Seq and qRT-PCR experiments always occurred. Additionally, the qRT-PCR results based on specific gene primers should be more reliable.Quantitative real-time PCR (qRT-PCR) was conducted to validate some of the DEGs. Nineteen genes were selected according to the following criteria: (a) significantly differential expression among different gall-formation stages and (b) previously reported to be potentially involved in a plant defense response such as genes involved in transcription factors, signaling, responses to stress, and secondary metabolism. Results of the qRT-PCR analysis were globally consistent with RNA-seq data . Only thD. kuriphilus is one of the most damaging pests of Castanea spp. [D. kuriphilus galling prevents flower and shoot development, and can even contribute to tree mortality [D. kuriphilus on Chinese chestnuts based on the transcriptome approach. As shown in nea spp. . D. kuriortality . Here, wEurosta solidaginis Fitch) and a gelechiid caterpillar , do not induce JA in their Solidago host plants [Mayetiola destructor Say) attack wheat (Triticum aestivum L.) [Daktulosphaira vitifoliae infestations [bHLH transcription factors were significantly upregulated according to RNA-Seq and qRT-PCR results  play a central role in this pathway ,22. Howet plants , nor doeivum L.) . In contstations . Additiostations . In the  results , suggest results . All of SA is another plant hormone reported to be involved in host responses to gall formation ,25. Cons2+ signaling was also involved in the plant response to biotic stress [2+ signaling can begin when plants respond to biotic stress [2+ signaling is involved in the plant defense responses to the bean pyralid (Lamprosema indicata F.) in highly resistant soybean [2+ signaling could play different roles depending on the level of resistance of the plant host to pests. For example, Ca2+ signaling can play a negative role in the regulation of defense responses in highly susceptible plant material [2+ [CML genes in soybeans are modulated in response to bean L. indicata larvae [CML genes were downregulated at stage A of the D. kuriphilus invasion on the chestnut but upregulated at growth stage B . Considering these results, we propose that cuticle metabolism might be affected by D. kuriphilus infestations and increase chestnut susceptibility in the initiation and growth stages. Among the genes related to cutin and wax metabolism, we identified cytochrome P450 with more upregulation after D. kuriphilus infestation. CYP has an important role in the defense of organisms against pests [D. kuriphilus. KEGG enrichment analysis showed that the secondary metabolic pathway began to show enrichment at stage A  [D. kuriphilus. Hence, we assumed that HR might also exist in C. mollissima BL and that HR might be triggered to increase chestnut resistance against D. kuriphilus.Plant defensive responses can be manipulated and reduced by the gall-inducing insects . When atcrenata) . RBOHD-dcrenata) . In this2O2 molecules during rice–gall midge interaction [2O2 in chestnut. In contrast, SOD genes in this study were almost downregulated. SOD is responsible for catalyzing the dismutation (or partitioning) of the superoxide (O2−) radical into either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2). Therefore, the decrease in SOD might also contribute to the decreased content of H2O2 in chestnuts during gall formation. Laloi et al. reported that the overexpression of ascorbate peroxidase (H2O2 scavenger) enhanced the intensity of the stress response that was mediated by a singlet oxygen in Arabidopsis [2O2 but by other ROS, and this is consistent with the rice–gall midge interaction [SOD and CAT, peroxidase has also been reported to be associated with plant responses to insects [D. kuriphilus  plants ‘Hongli’ were planted in Chinese chestnut germplasm resources, Nanjing City, Jiangsu Province, China , in April 2017. Leaf samples together with gall were collected at four gall stages (Initiation (A) at April 7, Early growth (B) at April 10, Late growth (C) at April 15, and Maturation (D) at April 26) in 2017. A healthy leaf was used as a control. Each sample contained three biological replicates. The fresh leaves were harvested, frozen in liquid nitrogen, and stored at −80 °C until use.One-year-old Chinese chestnut . The quantity and quality of the RNA samples were verified using a NanoDrop2000 Spectrophotometer , agarose gel electrophoresis (1.0%), and an Agilent 2100 Bioanalyzer . Satisfactory RNA with an RNA Integrity Number (RIN) greater than 7.0 was used for library construction and sequencing. The detailed information for total RNA quality and concentration was shown in C. mollissima Bl. reference genome (https://www.hardwoodgenomics.org/Genome-assembly/1962958) by using HISAT2 software [Clean data were obtained from raw data by removing adapter sequences and trimming reads with poly-N and low quality reads . Clean reads from all samples were mapped to a software  for furtThe NOISeq method was used to obtain the “base mean” value for identifying DEGs . Foldchahttp://www.ncbi.nlm.nih.gov/), Swiss-Prot database (http://www.expasy.ch/sprot), the COG (http://www.ncbi.nlm.nih.gov/COG/), Pfam (http://pfam.xfam.org/) databases, by using BLASTX (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with an E-value of 1e-5, and searched against the Pfam database (http://pfam.xfam.org/) by hmmscan (v3.0) software [p < 0.01 and FDR < 0.05 by agriGO [p < 0.05) and Benjamini (FDR < 0.05) were performed to detect statistically significant enrichment of KEGG pathways. GO and KEGG enrichment analyses were performed using the whole eggplant transcriptome setting as the background. Transcription factors were identified using PlantTFDB 4.0 with default parameters [https://github.com/raivokolde/pheatmap). The gene expression levels were transformed by log2 (mean of FPKM+1) using three biological replications.All of the mapped genes were compared with public databases, including the NR database . Detailed information on the genes and primer sequences are listed in Total RNA was isolated as described above. The first-strand cDNA was synthesized from 2 μg of total RNA using a Prime Script RT Reagent Kit . The qRT-PCR reactions were performed in 96-well plates using the ABI7500 fast Real-Time PCR system  with the SYBR Premix ExTaq™ Kit . The n stages . The relt method . Three bAll experiments were conducted with three biological replicates. Data analyses were conducted using SPSS version 17.0 statistical software.D. kuriphilus infestations via transcriptomic analysis of DEGs during different gall-formation stages. In response to insect attack, plant hormone signaling, transcription, and Ca2+-mediated signal transduction pathways, secondary metabolism and stress responses occurred in the Chinese chestnut. DEGs involved in these biological processes might represent Chinese chestnut defense genes. The study data provides new information on important molecular level pathways used by C. mollissima during D. kuriphilus gall formation.We investigated the molecular basis of Chinese chestnut responses to


P=0.17; MS HR: 0.99, 95% CI: 0.92-1.07, P=0.85); however, a weak, positive association was observed for GrimAgeAccel and invasive breast cancer . Stratification of invasive cancers by menopause status at diagnoses revealed the association was predominantly observed for postmenopausal breast cancer . Although the MS was unrelated to breast cancer risk, we find evidence that GrimAgeAccel may be weakly associated with invasive breast cancer, particularly for women diagnosed after menopause.Measures derived using blood DNA methylation are increasingly under investigation as indicators of disease and mortality risk. Three existing epigenetic age measures or “epigenetic clocks” appear associated with breast cancer. Two newly-developed epigenetic mortality predictors may be related to all-cancer incidence, but associations with specific cancers have not been examined in large studies. Using HumanMethylation450 BeadChips to measure blood DNA methylation in 2,773 cancer-free women enrolled in the Sister Study, we calculated two epigenetic mortality predictors: ‘GrimAgeAccel’ and the ‘mortality score’ (MS). Using Cox proportional hazard models, neither GrimAgeAccel nor the MS were associated with overall breast cancer incidence (GrimAgeAccel hazard ratio [HR]: 1.06, 95% confidence interval [CI]: 0.98-1.14,  Age related changes to blood DNA methylation (DNAm) have been extensively reported –4 and seWhile epigenetic clocks were designed to predict the biological consequences of aging, other combinations of CpGs have been identified to predict mortality , 27. GriGrimAgeAccel was previously reported to be associated with all-cancer incidence . Using DThe ‘mortality score’ (MS), a separate DNAm-based predictor, was also designed to predict all-cause mortality . Using 5Although GrimAgeAccel and the MS were designed to predict all-cause mortality, they may be useful in predicting cancer incidence. In additional to being strongly associated with all-cancer incidence, GrimAgeAccel may also predict menopause timing ; the MS P< 0.001), an age-adjusted MS was derived by regressing the MS on chronological age and predicting the residuals. The calculated residuals represent a MS that is independent of chronological age; as expected, this score and GrimAgeAccel were not correlated with chronological age  (P< 0.001)  had an average age of 56.6 years (standard deviation [SD]= 8.8 years); 70% of whom reported being postmenopausal. The women who developed breast cancer tended to be older at enrollment, drank more alcohol, were less physically active, had later ages at menopause, and were more likely to report postmenopausal hormone use . DistribP= 0.61) . However< 0.001) .in situ [DCIS] combined), in unadjusted models, neither of the epigenetic mortality predictors were associated with breast cancer incidence  (P=0.08) (P=0.41) (P=0.65) and neither the GrimAgeAccel nor age-adjusted MS metrics were associated with DCIS   . In anal(P=0.08) . However(P=0.41) . The age P=0.61) .P=0.04; age-adjusted MS HR: 0.99, 95% CI: 0.91, 1.08, P=0.82) (P=0.09) (P=0.40). No meaningful associations were observed for either epigenetic mortality predictor and invasive ER-negative tumors  . Account P=0.82) . Neither P=0.82) . A weak,(P=0.09) ; model ae tumors .P<0.001); cystatin C ; growth differentiation factor-1 ; leptin ; and tissue inhibitor metalloproteinase 1 . After adjustment for breast cancer risk factors, associations remained for the DNAm-predictors of cystatin C  and leptin . Similar patterns were observed for invasive breast cancer, although associations with DCIS tended to be weaker.As a post-hoc analysis to further interrogate the GrimAgeAccel metric, the separate DNAm-predictors that comprise DNAm GrimAge were tested with breast cancer overall and stratified by invasive cancers and DCIS. These components are DNAm-based predictors of circulating plasma protein concentrations, rather than directly measured concentrations. In unadjusted models, five DNAm GrimAge components were associated with breast cancer . SpecifiUsing a nationwide, prospective cohort designed to identify novel breast cancer risk factors, we found a weak association between GrimAgeAccel and invasive breast cancer. Further stratification of invasive breast cancers by tumor ER status and menopause status at diagnosis revealed somewhat enhanced associations between GrimAgeAccel and incidence of ER-positive and postmenopausal breast cancers. Although our findings suggest that this epigenetic mortality predictor may be a weak marker for these specific breast cancer subtypes, in post-hoc analyses, we found that some of the individual components of DNAm GrimAge appeared to have stronger associations with breast cancer. These components are DNAm-based predictions of circulating plasma proteins and are not based on direct measurements in study subjects. Despite the moderate correlation with GrimAgeAccel, the MS was not related to breast cancer incidence.Although GrimAgeAccel and the MS were both designed to predict all-cause mortality, we only observed breast cancer associations with GrimAgeAccel. Differences in the design of these two mortality predictors may explain the different associations: unlike the MS, DNAm GrimAge did not directly select mortality-associated CpGs. Instead, DNAm GrimAge is based on CpGs that are predictive of mortality-associated risk factors, specifically, smoking pack-years and various plasma proteins . InteresBoth epigenetic mortality predictors include CpGs previously reported to be associated with past smoking behaviors , 27, a sWhile we focused on unadjusted association estimates for the epigenetic mortality predictors, we found that adjustment for breast cancer risk factors attenuated associations. Although unadjusted analysis is appropriate given that we were primarily interested in assessing predictive utility of the epigenetic mortality predictors, the attenuation of the associations suggests the GrimAgeAccel metric may only be a marker of breast risk factors, rather than having independent associations with disease risk. Conversely, associations for the DNAm-predictors of cystatin C and leptin were robust to adjustment and may be useful DNAm-based markers for predicting breast cancer. Notably, in the FHS where these markers were developed, the DNAm-predictors of these proteins only had moderate correlations with directly measured concentrations  . AnotherIn summary, we find that the GrimAgeAccel mortality predictor was weakly associated with postmenopausal and ER-positive invasive breast cancers. Given the reliance on smoking-associated CpGs, it is perhaps unsurprising that the associations with breast cancer incidence were weak. Other components of DNAm GrimAge, however, appeared to be useful in predicting breast cancer. Blood DNAm may be a useful matrix to derive novel breast cancer predictors. For example, epigenetic clocks were designed to capture age effects and are associated with breast cancer incidence . Single in situ), and 1,479 additional women who developed breast cancer after blood draw (data release 6.0). Whole blood samples and informed consent were obtained at a home visit. The institutional review boards at the National Institute of Environmental Health Sciences and the Copernicus Group approved the study.The Sister Study is a prospective cohort of 50,884 cancer-free women, recruited from the United States (including Puerto Rico) and enrolled between 2003–2009 . EligiblProcessing procedures for the DNA samples have been previously reported . GenomicEnmix R software package was used for methylation data preprocessing and quality control [https://sisterstudy.niehs.nih.gov/English/coll-data.htm. GrimAgeAccel was calculated using an online calculator (https://dnamage.genetics.ucla.edu/home) and a continuous version of the MS was calculated as described by the developers [The  control . This in control . Data frvelopers .P-values. We treated chronological age as the time-scale in all models. For the primary analysis, we combined invasive and ducal carcinoma in situ to represent breast cancer overall. In secondary analyses, we considered those categories separately. We also investigated associations for invasive breast cancer by menopausal status at diagnosis and tumor estrogen receptor status. Because we were interested in assessing predictive utility of these biomarkers, we focused on unadjusted associations. However, we also examined associations accounting for established breast cancer risk factors, including: body mass index (BMI), menopause, a BMI-menopause interaction term, physical activity, alcohol intake, parity, age at first birth (among parous), age at menarche, breastfeeding duration, and hormone therapy and oral contraception duration [Although GrimAgeAccel was designed to be independent of chronological age, the MS was not. We therefore regressed the MS on chronological age and predicted the residuals to create a MS that was independent of chronological age to use in our main analyses. We assessed Pearson correlations between the epigenetic mortality predictors and chronological age. We standardized the epigenetic mortality predictors and the individual DNAm GrimAge components to have means of zero and standard deviations of one. To examine associations with breast cancer risk, we used case-cohort Cox proportional hazard models to calculate hazard ratios, 95% confidence intervals and duration , 53–57. Supplementary FiguresSupplementary Tables


Pelophylax esculentus complex, for the emergence of asexual reproduction. Further, we assessed the impact of its asexual reproduction on the maintenance of interspecies hybrids from two populations on the western edge of the P. esculentus range, in which hybrids coexist with either both parental species or with only one parental species. After analysing tadpole karyotypes, we conclude that in both studied populations, the majority of diploid hybrid males produced haploid gametes with the P. ridibundus genome after elimination of the P. lessonae genome. Hybrid females exhibited problems with genome elimination and duplication; they usually produced oocytes with univalents, but there were observations of individual oocytes with 13 bivalents and even 26 bivalents. In some hybrid tadpoles, especially F1 crosses, we observed failed germ cell development, while in tadpoles from backcrosses, germ cells were normally distributed and contained micronuclei. By identifying chromosomes present in micronuclei, we estimated that the majority of tadpoles from all crosses were able to selectively eliminate the P. lessonae chromosomes. According to our results, hybridogenesis in hybrids can appear both from crosses of parental species and crosses between sexual species with hybrid individuals. The ability to eliminate a genome and perform endoreplication to ensure gamete formation differed between male and female hybrids from the studied populations. Some diploid hybrid females can rarely produce not only haploid gametes but also diploid gametes, which is a crucial step in the formation of triploid hybrids.Many closely related species are capable of mating to produce hybrid offspring, which are usually sterile. Nevertheless, altering the gametogenesis of hybrid offspring can rescue hybrids from sterility by enabling asexual reproduction. Hybridogenesis is one of the most complicated asexual reproductive modes, and it includes drastic genome reorganization only in the germline; this is achieved through elimination of one parental genome and duplication of the remaining one to restore diploid chromosomal set and overcome blocks in meiotic progression. We investigated a model of hybridogenesis, namely, water frogs from the  Only those germ cells that successfully eliminated only P. lessonae chromosomes can divide further and produce gametes.Significant sterility and gametogenetic aberrations in the studied tadpoles and adult frogs can be related to abnormal genome elimination, which occurs in gonial cells. Moreover, the presence of univalents in oocytes and spermatocytes as well as frequently observed aneuploid, di- and polyploid gametes in hybrids from different populations also indirectly indicates the absence of genome elimination and endoreplication in hybrids ,30,45,46P. lessonae genome elimination, revealing a crucial step in establishing hybridogenetic reproduction. We suggest that initially, studied R-L-E systems can emerge after contact of P. ridibundus and P. lessonae . However, in such populations, hybrids usually produce gametes with P. ridibundus genome allowing them to reproduce only when they cross with P. lessonae individuals. Thus, here we confirm two different mechanisms of hybrid appearance in R-L-E systems: from primary crosses of parental species and from crosses of hybrid individuals with P. lessonae.Asexual hybrids can influence speciation in several ways. Asexual hybrids can not only restrict gene flow between parental species because recombination is prevented between their genomes but also mediate speciation themselves –20,47. IP. esculentus is extremely broad and almost ubiquitously coincides with the range of parental species. Nevertheless, in some localities, crosses of P. ridibundus with P. lessonae do not always lead to the appearance of viable and fertile P. esculentus. This can be explained by high levels of polymorphisms that have been previously described between different populations of P. ridibundus and P. lessonae [P. ridibundus related form P. cf. bedriagae can also affect the possibility of genome elimination [P. ridibundus but rarely observed in hybrids [P. ridibundus can induce hybridogenesis after crosses with P. lessonae or several lessonae-related species, while even the closely related Balcan taxon P. kurtmuelleri seems unable to produce asexual hybrids [Our results partially support the hypothesis of an independent origin of hybridogenetic hybrids in studied population systems. However, the distribution of lessonae ,16,51. Mmination . Such in hybrids . This is hybrids .P. esculentus can either displace parental P. ridibundus or invade habitats that are suitable for P. lessonae, leading to the appearance of L-E systems. We found that hybrid males from both L-E and R-L-E systems produced hybrid tadpoles via the formation of gametes with the P. ridibundus genome . Our results are in close agreement with earlier data about sperm genome composition in P. esculentus males from studied locations [P. ridibundus genome by diploid hybrid males was widely observed in different L-E systems located in western Russia, Poland, Germany, Slovakia, Latvia, and Belarus [P. ridibundus mtDNA in hybrid females (11%) in some L-E systems indicates that individual hybrid females are able to reproduce [P. ridibundus gametes.Taking into account the similarity in mechanisms of maintaining L-E and R-L-E systems in the Mari El Republic, we suggest their common origin in the studied hybrid complexes. Hybridogenetic ocations ,26. The  Belarus ,47,51,53The achievement of sexual reproduction via the formation of polyploid animals, in particular triploids, which are also known as the “triploid bridge” is an important stage in the hybrid speciation concept , 54, 55.P. ridibundus chromosomes; thus, such tadpoles are able to eliminate the genome selectively.This study demonstrates a notable link between cytological mechanisms causing asexuality and their impact on hybrid populations and speciation. We found that asexual reproduction is not always achievable for hybrid animals, since some tadpoles exhibit abnormal development of germ cells and eliminate chromosomes of both parental species with variable rates of selectivity. However, even in F1 crosses, some hybrid tadpoles form micronuclei that preferentially include . We found that in R-L-E systems, hybridogenetic P. esculentus can emerge from crosses of parental species and from crosses of P. esculentus males with P. lessonae females. Surprisingly, hybrid reproduction in both R-L-E and L-E systems depends basically on P. esculentus males producing gametes with P. ridibundus genome but not on P. esculentus females. Hybrid females in the investigated systems exhibit numerous abnormalities in genome elimination and/or endoreplication, leading to prevalent formation of oocytes with 26 univalents. However, some hybrid females produced oocytes with 13 bivalents of P. ridibundus as well as rare oocytes with 26 bivalents, which are subsequently able to give rise to triploid hybrids. Thus, populations from the western border of P. esculentus range in the Mari El Republic represent an example of the initial stages of speciation via interspecies hybridization.Based on our results, we propose a model of hybrid frog reproduction in the studied R-L-E and L-E systems S1 TableP. lessonae individuals are marked in red, crosses of P. lessonae with diploid hybrids from L-E systems are marked in blue and those with R-L-E systems are marked in green. Frogs used for lampbrush chromosome analysis from L-E systems (marked blue) and R-L-E systems (marked green).Crosses of parental individuals are marked in orange, crosses between (PDF)Click here for additional data file.S2 TableP. lessonae individuals (marked red) and P. lessonae individuals and hybrids from L-E systems (marked blue) and R-L-E systems (marked green).Tadpoles obtained from crosses of parental individuals (marked orange), two (PDF)Click here for additional data file.S1 FigP. ridibundus chromosomes; another 13 uni- or bivalents do not have a signal in the centromeric region, thus corresponding to P. lessonae chromosomes. The chromosomal set represented in S1A Fig corresponds to Fig 3B. The chromosomal set represented in S1B Fig corresponds to Fig 3C.Lampbrush chromosome sets, including 26 univalents (a) and 26 bivalents (b), among which 13 uni- or bivalents have a signal in the centromeric region, thus corresponding to (PDF)Click here for additional data file.


Helicobacter pylori and cardiovascular disease have yielded variable results and no studies have been conducted in nonhuman primates. In this investigation, we examined the relationship between H. pylori infection and atherosclerosis development in socially housed, pre- and postmenopausal cynomolgus macaques consuming human-like diets. Ninety-four premenopausal cynomolgus monkeys (Macaca fascicularis) were fed for 36 months an atherogenic diet deriving its protein from either casein lactalbumin(CL) or high isoflavone soy (SOY). Animals were then ovariectomized and fed either the same or the alternate diet for an additional 36 months. Iliac artery biopsies were obtained at the time of ovariectomy and iliac and coronary artery sections were examined at the end of the study. Evidence of H. pylori infection was found in 64% of the monkeys and 46% of animals had live H. pylori within coronary atheromas as determined by mRNA-specific in situ hybridization. There was a significant linear relationship between the densities of gastric and atheroma organisms. Helicobactor pylori infection correlated with increased intimal plaque area and thickness at both the premenopausal and postmenopausal time points and regardless of diet (p< 0.01), although animals consuming the SOY diet throughout had the least amount of atherosclerosis. Additionally, plasma lipid profiles, intimal collagen accumulation, ICAM-1, and plaque macrophage densities were adversely affected by H. pylori infection among animals consuming the CL diet, while the SOY diet had the opposite effect. Plaque measurements were more highly associated with the densities of cagA-positive H. pylori within coronary atheromas than with the densities of gastric organisms, whereas plasma lipid changes were associated with H. pylori infection, but not cagA status. This study provides strong evidence that live H. pylori infects atheromas, exacerbates atherosclerotic plaque development, and alters plasma lipid profiles independently of diet or hormonal status. Finally, socially subordinate animals relative to their dominant counterparts had a greater prevalence of H. pylori, suggesting a stress effect. The results indicate that early H. pylori eradication could prevent or delay development of cardiovascular disease.A number of viruses and bacterial species have been implicated as contributors to atherosclerosis, potentially providing novel pathways for prevention. Epidemiological studies examining the association between  Chlamydia pneumoniae, and H. pylori . Br. Br38]. Total plasma cholesterol (TPC), high-density lipoprotein-associated cholesterol (HDLc), and plasma triglycerides (TGs) were determined on numerous occasions as well as at the end of the study by the Wake Forest Primate Center Clinical Chemistry Laboratory , 43, whiSerum from each animal was submitted for viral detection. Antibodies against Herpes B virus, simian immunodeficiency virus (SIV), measles, and simian T cell leukemia virus (STLV-1 and -2) were assessed by ELISA. Rhesus cytomegalovirus (RhCMV), simian type D retrovirus (SRV/D), and STLV-1 and-2 were detected by immunofluorescence assay .H. pylori. Due to postmortem degradation, usable gastric tissue could not be obtained from three animals. Coronary artery tissue from these animals was included in the analysis only if H. pylori was detected at that site, since lack of H. pylori in the artery does not guarantee that the animal was H. pylori-negative in the stomach.At the conclusion of the study, surviving monkeys (n = 74) were euthanized using sodium pentobarbital  and perfused with lactated Ringer’s solution. A complete necropsy was performed and all major organs, including the stomach, were collected and fixed in 10% neutral buffered formalin (NBF). The right common iliac artery (RCI) was removed for comparison with the left common iliac artery that was removed at the time of ovariectomy. Hearts were removed and coronary arteries were perfused-fixed with 10% NBF for 1h at 100 mm Hg pressure. The left anterior descending (LAD), left circumflex (LCX), and right coronary arteries (RCA) were then collected. Each artery was sectioned into five blocks (3 mm each in length), cut perpendicular to the long axis of the artery, processed routinely, and embedded in paraffin to generate formalin-fixed paraffin-embedded (FFPE) blocks. Whole sections of the left circumflex artery were also sent to the Uniformed Services University of the Health Sciences for detection of H. pylori. Slides were examined by light microscopy for the presence/absence of argyrophilic curved or spiral-shaped bacteria. The remaining slides were used for in situ hybridization as detailed below.For each animal, the first 5mm FFPE block of the LCX (n = 74) was serially sectioned at 5um thickness and sections were mounted on slides. The first five sections were stained with Verhoeff -van Gieson stain to highlight the internal elastic lamina, and plaque size was determined using computer assisted morphometric analysis (ImageProPlus 5.1); the cross-sectional area of plaque  in each of the five sections was measured and plaque area for each animal was calculated as the mean of the five sections examined. Section 6 was stained with Hematoxylin-Eosin (H&E), examined by light microscopy for the presence of mineralization and/or necrosis within the tunica intima, and semi-quantitatively scored as follows: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. Section seven was stained with Masson’s Trichrome to highlight areas of collagen deposition (fibrosis). The proportion of intima occupied by fibrosis was determined by overlay of a 25 x 25 pixel grid with evaluation of each crosshatch as positive or negative for collagen by a board-certified pathologist (JAC) blinded to treatment. The remaining LCX serial sections were utilized for immunohistochemistry as detailed below. Additional 5 μm sections of stomach tissue and LCX artery were generated, mounted on slides and stained with H&E and Genta-Robason , which is a conventional method for detecting the presence of For immunohistochemistry, the slides were incubated with mouse anti-human HAM56 , mouse anti-human CD3 , mouse anti-human CD4 , and mouse anti-human CD8  for 1.5 h at 25° C. Primary antibodies were localized with appropriate biotinylated secondary antibodies, streptavidin–alkaline phosphatase  and Vector Red  substrate. Sections were counterstained with Mayer's Hematoxylin and examined by the study pathologist (JAC) using light microscopy. Thymus and lymph node were used as tissue controls; as an assay control, the primary antibodies were substituted with non-immune sera. Cell densities for macrophages, CD3+ cells, CD4+ cells, and CD8+ cells were determined by overlay of a 25 x 25 pixel grid with evaluation of each crosshatch for positive or negative staining by the pathologist (JAC), who was blinded to treatment. Cell densities are expressed as the percentage of intima occupied by positive staining.–ΔCT procedure . H.eviously . These iGT AC-3’).In situ hybridization was performed as previously described [cagA mRNA. For fluorescence dual detection studies, 16S rRNA probes were labeled with biotin and detected using avidin-Texas red (red color), while cagA probes were labeled with digoxigenin-3-0-methylcarbonyl-ε-aminocaproic acid-N-hydroxy-succinimide ester  and detected using anti-digoxigenin-FITC (green color).escribed , 54, 55.2, i.e. 17,892 μm3 for 5 μm-thick sections; No. KR-409 , located in a Nikon Eclipse E 800. Tissues were analyzed randomly and blindly regardless of category. All data were expressed as mean ± standard error of the mean (SEM) number of bacteria per 106 μm3 (representing an imaginary cube with sides of 100 μm or 0.1 mm).An Eclipse Nikon microscope was used to examine the section at X100, X400 and X1000 magnification for bright light and for fluorescence. Bacterial density was quantified as described previously . BrieflyH. pylori densities, cagA densities, H. pylori infection status, and social status. Linear models and analysis of variance (ANOVA) were used to determine the influences of H. pylori infection, diet, and social status on severity of premenopausal and postmenopausal atherosclerosis, arterial inflammatory markers, and lipid profiles. Pairwise group comparisons were performed using Welch two-sample t-test and/or nonparametric rank tests , the latter employed when assumptions for the validity of the t-test were not satisfied. Linear models and correlation analyses were used to compare the effect of H. pylori density in the artery and stomach on plaque area and thickness. Fisher’s exact test was used where indicated. The (effective) sample sizes in some of the calculations were smaller than the overall sample sizes due to missing values.Descriptive statistics and graphical plots, such as means, standard deviations and standard errors, and comparative boxplots, were calculated and obtained for H. pylori were first detected in gastric mucosa using the Genta-Robason staining procedure. Subsequently live H. pylori were specifically detected in gastric mucosa and LCX coronary artery sections by in situ hybridization using RNA anti-sense probes specific for H. pylori 16S rRNA or cagA mRNA. These probes recognize RNA and the method has been previously validated [H. pylori is for the majority of organisms to reside in the mucus just above the epithelium, attached to epithelial cells, or within gastric crypts. H. pylori is occasionally intracellular or found within gastric capillaries[H. pylori were located in the superficial gastric mucosa  . When the densities of H. pylori in the coronary arteries were plotted against gastric mucosal densities, a substantial linear correlation  was found  with densities in the stomach. In other words, live H. pylori were frequently present in coronary arteries of H. pylori-infected monkeys and the density of bacteria found in plaques was strongly related to colonization density in the stomach.Of potentially greater interest, lication . Using s (green) . Occasio (green) , but theas found . A correH. pylori infection  was evident at both premenopausal and postmenopausal time points. Premenopausal atherosclerosis was assessed in the LCI at the time of ovariectomy and postmenopausal atherosclerosis was assessed in both the RCI and coronary arteries . Both the intimal area (IA) and maximum intimal thickness (MXIT) were strongly associated with H. pylori infection (p<0.005) even when diet was included in the statistical model . There was no significant association between H. pylori infection status and premenopausal estrogen receptor expression. Other premenopausal measurements shown in The influence of al model . Few H. H. pylori-infected animals, but progression of plaque thickness was unaffected by infection status.As shown in cagA-positive H. pylori. Large necrotic regions and bulging plaques in the LCX were found in six of the cagA-positive animals and none of the cagA-negative or uninfected animals (Fisher’s exact test p< 0.05). Plaques representative of median thickness values for each cagA-positive, cagA-negative, and uninfected macaques are shown in H. pylori density was determined, not the maximum thickness of the entire artery section. Plaque measurements for the three coronary arteries were averaged and the differences between H. pylori-positive and–negative animals irrespective of diet group are shown in H. pylori infection substantially increased intimal area and thickness . Panels 10C and 10D demonstrate the effects of both H. pylori infection and diet group. H. pylori-negative animals fed a SOY-SOY diet had essentially no plaque accumulation, whereas several H. pylori-positive animals on the same diet had modest amounts of plaque. Almost all H. pylori-positive animals that received a casein/lactalbumin diet at any point had moderate to severe atherosclerosis. Analyses of the effects of diet on atherosclerosis in these animals have been previously published [The micrographs in ublished , 44, andcagA-positive animal receiving the casein-lactalbumin diet for the duration of the study. There is no a priori basis for invalidating the measurements from this animal. Moreover, the results of analyses were similar whether the outlier was included or not, and all statistical analyses discussed herein are inclusive of all animals.The outlier seen in H. pylori on plaque measurements were due to local effects of bacteria within the plaques or due to systemic effects stemming from gastric infection. To this end, we performed multiple regression analysis and correlation analysis. As shown in cagA-positive H. pylori in the LCX coronary artery showed a stronger correlation to plaque area and thickness than did the density of cagA-positive bacteria in the stomach. On the other hand, the overall densities of H. pylori, as measured by 16S in the stomach or artery, did not correlate with plaque measurements , and increased TPC/HDLc (Castelli’s ratio)  correlated better with plasma lipids than did H. pylori density in the artery. Also, the presence of cagA did not appear to be an important contributor to dyslipidemia, as indicated by the stronger correlations between plasma lipids and stomach H. pylori 16S than plasma lipids and artery or stomach cagA. This suggests that either local effects of H. pylori in the stomach or systemic effects of infection are responsible for plasma lipid alterations. Body weight, total cholesterol, and triglycerides were not associated with H. pylori infection.dication , 58. SimHuman epidemiological studies have noted a relationship between atherosclerosis and viral infections . To explH. pylori infection influence this risk? We found that subordinate status was significantly associated with increased risk for H. pylori infection and increased infection density (p< 0.05 using either Fisher’s exact test or Pearson’s Chi-squared test with Yates’ continuity correction). Only 48% of the dominant monkeys were infected with H. pylori as compared with 78% of the subordinate animals; however, rank did not influence either premenopausal or postmenopausal plaque measurements when H. pylori infection status was included in the predictive model. Therefore, H. pylori infection may be a more important contributor to atherosclerosis than social status or, alternatively, status may exert its effects in part through modified resistance to this bacterial infection.Premenopausal female cynomolgus macaques of low social status are typically at greater risk of atherosclerosis than their dominant counterparts . Could HH. pylori on development of atherosclerosis in non-human primates and the first to demonstrate that H. pylori within atherosclerotic plaques are alive. Natural infection with H. pylori has been documented in both rhesus (M. mulatta) and cynomolgus macaques [H. pylori gastric pathogenesis, evolution, and carcinogenesis [H. pylori to influence extragastric pathology in non-human primates had not been explored previously.This work is the first study to examine the effects of macaques –62, and ogenesis , 63, 64.H. pylori DNA by PCR in human atherosclerotic plaques was first reported by Farsak et al. in 2000 and has since been confirmed by a number of other investigators [H. pylori within plaques. In situ hybridization has been routinely used to detect mRNA in paraffin-embedded tissue and is a sensitive and specific method for detecting H. pylori in gastric biopsies [cagA mRNA, combined with corresponding structures matching the size and morphology of H. pylori, confirm that live H. pylori inhabit atherosclerotic plaques. The presence of bacterial clusters further suggests that limited bacterial growth occurs within these same plaques. Seeding of heart valves by hematogenously spread oral flora is a well-documented risk and H. pylori within gastric and oral blood vessels has been previously documented [H. pylori entering the bloodstream can reach the heart and adhere. H. pylori requires host cholesterol for optimal membrane integrity [H. pylori. The distinction between living or dead H. pylori within plaques is critical because live H. pylori can inject bacterial components including the CagA toxin, peptidoglycan, DNA, and metabolites of lipopolysaccharide into the host cell cytoplasm via the type IV secretion apparatus [H. pylori cannot influence host cell signaling in this manner. Therefore, in terms of atherosclerosis H. pylori is likely to directly activate inflammatory pathways within the atheroma in addition to acting indirectly via distal activity at the site of gastric colonization.Detection of tigators –29; nonebiopsies , 66. Ourcumented , 67, so ntegrity , 69 and pparatus , 71; deaH. pylori significantly worsened several plaque parameters, including common iliac artery intimal thickness and area in the same animals evaluated pre- and postmenopausally. Intimal collagen density and mineralization were also affected. Although the majority of macaques were infected with a mixture of cagA-positive and cagA-negative H. pylori strains, we were able to examine the contributions of cagA by determining whether LCX artery measurements were more greatly influenced by total bacterial density (as determined by 16S) or by cagA-positive bacterial density. That analysis revealed that cagA-positive strains were more atherogenic than cagA-negative strains. Moreover, H. pylori densities found within the arteries had a greater impact than bacterial density in the stomach. Though not conclusive, these data suggest that plaque development is due to local effects of cagA-positive H. pylori. Notably, these findings are consistent with epidemiological studies conducted in humans. While early epidemiological studies investigating the role of H. pylori in cardiovascular diseases yielded mixed results [cagA-positive vs. cagA-negative strains have yielded more consistent positive associations [et al reported cross-reactivity between CagA-specific antibodies and artery tissue [H. pylori proteins reacted with the tissue or prove that the “cross-reactive” antigen was of human origin. In light of the evidence shown here, it is likely that CagA itself was being detected. results –20, receciations , 72–74. y tissue . This imH. pylori in the gastric mucosa were highly correlated with the densities in the coronary artery plaques. Gastric colonization density is known to vary among individuals and is believed to be related to both the H. pylori strain and the type of immune response generated by the host [H. pylori densities in both locations change over time and whether gastric biopsies could be used to estimate the risk of severe atherosclerosis.Another intriguing finding of this study was that the densities of the host –78. We cH. pylori infection was associated with increases in some arterial inflammatory markers, providing a possible mechanism for H. pylori-mediated atherosclerosis exacerbation. VCAM-1, ICAM-1, and MCP-1 all contribute to recruitment of macrophages to plaques [H. pylori-infected macaques. IL-6, which induces ICAM-1 [H. pylori. Our findings are consistent with in vitro induction of P-selectin, E-selectin, VCAM-1, and ICAM-1 and enhanced transendothelial migration of activated CD4+ and CD8+ T cells in human umbilical vein endothelial cell cultures following H. pylori exposure [+ T cells, which are pro-atherogenic [H. pylori-positive animals. Others have reported that H. pylori induces MCP-1, IL-6, and ICAM-1 in various cell types [H. pylori DNA in human atherosclerotic plaques [et al. report that acute coronary artery disease patients infected with cagA-positive H. pylori have higher circulating IL-6 levels than uninfected patients [H. pylori within atherosclerotic lesions can directly interact with macrophages and/or endothelial cells to induce cytokine and adhesion molecule production.In addition to affecting plaque measurements,  plaques , 80 and s ICAM-1 , was alsexposure , 83. We erogenic , in H. pll types –88 and e plaques . Furtherpatients . Our resH. pylori, and some studies have found reversal of dyslipidemia following H. pylori eradication [H. pylori-associated dyslipidemia in male, but not female subjects [H. pylori-positive patients [H. pylori densities than arterial densities.Clinical studies have also reported decreased HDL and increased LDL in association with dication , 58, 91,dication . One Japsubjects ; we cleapatients , 95. ThiH. pylori increased atheroma measurements both pre- and post-menopausally and generally counteracted the beneficial effects of SOY on inflammatory markers in cynomolgus macaques ; howeveH. pylori burdens among dominant and subordinate animals have not been previously reported. Female subordinate macaques are under considerably more stress than dominant females and typically develop more atherosclerosis than their dominant counterparts when consuming an atherogenic diet [H. pylori prevalence, presumably due to poor hygiene and overcrowding [H. pylori [H. pylori burden in humans with low socioeconomic status could contribute to higher atherosclerosis risk in this group.The differing nic diet , an effenic diet . In humacrowding . Althougcrowding , subordicrowding . Moreove. pylori . Of note. pylori , the incH. pylori transmission occurs within 90 days via social contact, rather than fomites, but some animals remain uninfected, possibly due to host genetic factors [H. pylori infection persists for at least a decade in Rhesus macaques [H. pylori status and premenopausal plaque parameters further indicate that animals were infected before the study midpoint.The primary weakness of this study is that we are not certain when the animals became infected or whether bacterial prevalence changed over time. Humans most often become colonized during childhood, but transmission can occur later in life, particularly between spouses –103. In  factors , 105. H.macaques  indicatiH. pylori can reside within atherosclerotic plaques and that H. pylori is likely to have local effects on plaque size. Furthermore, H. pylori influenced macrophage infiltration, collagen deposition, ICAM1 expression, and plasma lipids. The effects of H. pylori on plaque parameters appeared to be primarily due to local effects of cagA-positive H. pylori, whereas changes in plasma lipids related to gastric infection by either cagA-positive or–negative strains. H. pylori status was relevant during both the premenopausal and postmenopausal phases as demonstrated by correlations between H. pylori infection and iliac artery plaque parameters measured at both ovariectomy and necropsy. Given the many similarities in atherogenesis between humans and non-human primates, future inclusion of cagA-positive H. pylori status in human epidemiological and clinical atherosclerosis studies is warranted.For the first time, this study conclusively demonstrated that live S1 Table(XLSX)Click here for additional data file.