To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant

Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.

Improving the accuracy of genomic prediction for meat quality traits using whole genome sequence data in pigs

Background Pork quality can directly affect customer purchase tendency and meat quality traits have become valu-able in modern pork production.However,genetic improvement has been slow due to high phenotyping costs.In this study,whole genome sequence(WGS)data was used to evaluate the prediction accuracy of genomic best linear unbiased prediction(GBLUP)for meat quality in large-scale crossbred commercial pigs.Results We produced WGS data(18,695,907 SNPs and 2,106,902 INDELs exceed quality control)from 1,469 sequenced Duroc×(Landrace×Yorkshire)pigs and developed a reference panel for meat quality including meat color score,marbling score,L*(lightness),a*(redness),and b*(yellowness)of genomic prediction.The prediction accuracy was defined as the Pearson correlation coefficient between adjusted phenotypes and genomic estimated breeding values in the validation population.Using different marker density panels derived from WGS data,accuracy differed substantially among meat quality traits,varied from 0.08 to 0.47.Results showed that MultiBLUP outperform GBLUP and yielded accuracy increases ranging from 17.39%to 75%.We optimized the marker density and found medium-and high-density marker panels are beneficial for the estimation of heritability for meat quality.Moreover,we conducted genotype imputation from 50K chip to WGS level in the same population and found average concord-ance rate to exceed 95%and r^(2)=0.81.Conclusions Overall,estimation of heritability for meat quality traits can benefit from the use of WGS data.This study showed the superiority of using WGS data to genetically improve pork quality in genomic prediction.

Breed identification using breed‑informative SNPs and machine learning based on whole genome sequence data and SNP chip data

Background Breed identification is useful in a variety of biological contexts.Breed identification usually involves two stages,i.e.,detection of breed-informative SNPs and breed assignment.For both stages,there are several methods proposed.However,what is the optimal combination of these methods remain unclear.In this study,using the whole genome sequence data available for 13 cattle breeds from Run 8 of the 1,000 Bull Genomes Project,we compared the combinations of three methods(Delta,FST,and In)for breed-informative SNP detection and five machine learning methods(KNN,SVM,RF,NB,and ANN)for breed assignment with respect to different reference population sizes and difference numbers of most breed-informative SNPs.In addition,we evaluated the accuracy of breed identification using SNP chip data of different densities.Results We found that all combinations performed quite well with identification accuracies over 95%in all scenarios.However,there was no combination which performed the best and robust across all scenarios.We proposed to inte-grate the three breed-informative detection methods,named DFI,and integrate the three machine learning methods,KNN,SVM,and RF,named KSR.We found that the combination of these two integrated methods outperformed the other combinations with accuracies over 99%in most cases and was very robust in all scenarios.The accuracies from using SNP chip data were only slightly lower than that from using sequence data in most cases.Conclusions The current study showed that the combination of DFI and KSR was the optimal strategy.Using sequence data resulted in higher accuracies than using chip data in most cases.However,the differences were gener-ally small.In view of the cost of genotyping,using chip data is also a good option for breed identification.

Low-depth whole genome sequencing reveals copy number variations associated with higher pathologic grading and more aggressive subtypes of lung non-mucinous adenocarcinoma

Objective:Histology grade,subtypes and TNM stage of lung adenocarcinomas are useful predictors of prognosis and survival.The aim of the study was to investigate the relationship between chromosomal instability,morphological subtypes and the grading system used in lung non-mucinous adenocarcinoma(LNMA).Methods:We developed a whole genome copy number variation(WGCNV)scoring system and applied next generation sequencing to evaluate CNVs present in 91 LNMA tumor samples.Results:Higher histological grades,aggressive subtypes and more advanced TNM staging were associated with an increased WGCNV score,particularly in CNV regions enriched for tumor suppressor genes and oncogenes.In addition,we demonstrate that 24-chromosome CNV profiling can be performed reliably from specific cell types(<100 cells)isolated by sample laser capture microdissection.Conclusions:Our findings suggest that the WGCNV scoring system we developed may have potential value as an adjunct test for predicting the prognosis of patients diagnosed with LNMA.

Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

ve To develop a reliable and sensitive method for detection of sex and multi-loci of Duchenne muscular dystrophy (DMD) gene in single cell

Gene expression in rat mesenchymal stem cells following treatment with natural cerebrolysin-containing serum Validation of a whole genome microarray technique

BACKGROUND： Natural cerebrolysin （NC）, a Chinese herbal drug for the treatment of Alzheimer＇s disease （AD）, induces mesenchymal stem cell （MSC） differentiation into neuron-like cells, with low toxicity. But the mechanisms involved in NC effects on MSCs remain poorly understood. OBJECTIVE： We used a whole genome microarray technique to further investigate the molecular, genetic, and pharmacodynamic mechanisms of NC on MSC gene expression profiles. DESIGN, TIME AND SETTING： A parallel, controlled, in vitro experiment was performed at the First Affiliated Hospital of Shenzhen University, Shenzhen Institute of Integrated Chinese and Western Medicine, China, between September 2006 and October 2008. MATERIALS： NC was provided by Shenzhen Institute of Integrated Chinese and Western Medicine China. It was predominantly composed of Renshen （Radix Ginseng）, Tianma （Rhizoma Gastrodiae） and Yinxingye （Ginkgo Leaf） and prepared by conventional water extractJon technology. Twelve adult, male, New Zealand rabbits were included, six of which underwent intragastric administration of NC extract for 1 month to create NC-containing serum. METHODS： Bone marrow was collected from the tibia and femur of Sprague Dawley rats, aged 6 8 months old. Rat MSCs were isolated and purified by the whole bone marrow adherence method. After in vitro culture, MSCs from passage 4 were treated with NC-containing serum for 48 hours, and total RNA was extracted. Gene expression in MSCs was analyzed using Affymetrix whole genome microarray analysis. MAIN OUTCOME MEASURES： Differentially expressed genes in NC serum-treated MSCs. RESULTS： NC treated MSCs displayed 46 differentially expressed genes, 22 with upregulated expression （fold change 〉 2） and 24 with downregulated expression （fold change 〈 -2）. Differentially expressed genes participated in neuronal growth, differentiation, and function, cell growth, differentiation, proliferation, apoptosis, signal transduction, substance/energy metabolism, ion transport, and immune responses. NC treatment changed levels of transforming growth factor β/ bone morphogenetic proteins, Hedgehog, Bmp, and Wntsignaling pathways, which regulate nerve cell differentiation, development and function, as well as learning and memory; Ras, G protein- coupled receptor signal pathways that are related to cell growth, proliferation, and apoptosis; and mitogen-activated protein kinase kinase kinase signaling cascades. CONCLUSION： NC can regulate gene expression for many signal transduction pathways related to nerve cell differentiation, development and function, learning and memory function, as well as regulation of cell growth, differentiation, proliferation, or apoptosis to mediate the genetic effects of NC treatment on AD.

Identification and characterization of long-InDels through whole genome resequencing to facilitate fine-mapping of a QTL for plant height in soybean(Glycine max L.Merr.)

With the development of sequencing technology, insertions-deletions(InDels) have been increasingly reported to be involved in the genetic determination of agronomical traits. However, most studies have focused on the identification and application of short-InDels(1–15 bp) for genetic analysis. The objective of this study was to deeply deploy long-InDels(>15 bp) for the genetic analysis of important agronomic traits in soybean. A total of 13 573 polymorphic long-InDels were identified between parents Zhongpin 03-5373(ZP) and Zhonghuang 13(ZH), which were unevenly distributed on 20 chromosomes of soybean, varying from 321 in Chr11 to 1 246 in Chr18. Consistent with the distribution pattern of annotated genes, the average density of long-InDels in arm regions was significantly higher than that in pericentromeric regions at the P=0.01 level. A total of 2 704(19.9% of total) long-InDels were located in genic regions, including 319 large-effect long-InDels, which resulted in truncated or elongated protein sequences. A previously identified QTL(qP H16) underlying plant height was further analyzed, and it was found that 26 out of 35(74.3%) long-InDel markers located in the qPH16 region showed clear polymorphisms between parents ZP and ZH. Seven markers, including three long-InDels and four previously reported SNP markers, were used to genotype 242 recombinant inbred lines derived from ZP×ZH. As a result, the qPH16 locus was narrowed from a 960-kb region to a 477.55-kb region, containing 65 annotated genes. Therefore, these long-InDels are a complementary genetic resource of SNPs and short-InDels for plant height and can facilitate genetic studies and molecular assisted selection breeding in soybean.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

Objective To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR （M-RTPCR）, for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing （NGS） platform. Methods Virus genome copy was quantified and seria（iy diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. Results The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing Iow-titer virus. Conclusion The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Biological Characterization and Whole Genome Sequencing of a Genotype VII Newcastle Disease Virus Strain

[Objective] The aim of this paper was to study the genotype, pathogenicity and nucleotide difference between Newcastle disease virus(NDV) isolates and traditional NDV vaccine strain(La Sota). [Method] A suspected NDV strain was isolated from a chicken farm. The isolate was preliminarily determined by HA and HI tests. A pair of primers was designed based on the partial sequence of NDV F gene published in GenBank(accession No. JF950510.1). F gene was amplified by RT-PCR, cloned and sequenced. The sequencing result was compared with the F gene sequences published in GenBank, and the phylogenetic tree was constructed to analyze the genotypes. The pathogenicity of the virus was determined by mean death time(MDT) of chicken embryos, intracerebral pathogenicity index(ICPI) of one-day-old chicks and intravenous pathogenicity index(IVPI) of six-week-old chickens, respectively. Based on the NDV genome sequence published in GenBank(accession No. JF950510.1), nine pairs of primers were designed to amplify the genome sequence of the isolate, and its structure was analyzed. [Result] The length of F gene was about 500 bp, and a NDV strain of genotype VII was isolated. The MDT, ICPI and IVPI were 52.8 h, 1.675 and 2.46, respectively, indicating the isolate was a virulent strain. The whole genome sequence analysis results showed that the full genome length of the isolate was 15 192 bp, which had 6 more nu-cleotides than that of La Sota strain, and the homology between the two strains was 82.8%. [Conclusion] A virulent NDV strain of genotype Ⅶ was isolated, with low homology to La Sota strain in nucleotide sequence.

Widespread Whole Genome Duplications Contribute to Genome Complexity and Species Diversity in Angiosperms

Gene duplications provide evolutionary potentials for generating novel functions, while polyploidization or whole genome duplication （WGD） doubles the chromosomes initially and results in hundreds to thousands of retained duplicates. WGDs are strongly supported by evidence commonly found in many species-rich lineages of eukaryotes, and thus are considered as a major driving force in species diversification. We per- formed comparative genomic and phylogenomic analyses of 59 public genomes/transcriptomes and 46 newly sequenced transcriptomes covering major lineages of angiosperms to detect large-scale gene dupli- cation events by surveying tens of thousands of gene family trees. These analyses confirmed most of the previously reported WGDs and provided strong evidence for novel ones in many lineages. The detected WGDs supported a model of exponential gene loss during evolution with an estimated half-life of approx- imately 21.6 million years, and were correlated with both the emergence of lineages with high degrees of diversification and periods of global climate changes. The new datasets and analyses detected many novel WGDs widely spread during angiosperm evolution, uncovered preferential retention of gene functions in essential cellular metabolisms, and provided clues for the roles of WGD in promoting angiosperm radiation and enhancing their adaptation to environmental changes.

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,

Functional Divergence between Subgenomes anc Gene Pairs after Whole Genome Duplications

Gene loss following whole genome duplication （WGD） is often biased, with one subgenome retaining more ancestral genes and the other sustaining more gene deletions. While bias toward the greater expression of gene copies on one subgenome can explain bias in gene loss, this raises the question to what drives differences in gene expression levels between subgenomes. Differences in chromatin modifications and epigenetic markers between subgenomes in several model species are now being identified, providing an explanation for bias in gene expression between subgenomes. WGDs can be classified into duplications with higher, biased gene loss and bias in gene expression between subgenomes versus those with lower, unbiased rates of gene loss and an absence of detectable bias between subgenomes; however, the origi- nally proposed link between these two classes and whether WGD results from an allo- or autopolyploid event is inconsistent with recent data from the allopolyploid Capsella bursa-pastoris. The gene balance hypothesis can explain bias in the functional categories of genes retained following WGD, the difference in gene loss rates between unbiased and biased WGDs, and how plant genomes have avoided being overrun with genes encoding dose-sensitive subunits of multiprotein complexes. Comparisons of gene expression patterns between retained transcription factor pairs in maize suggest the high degree of retention for WGD-derived pairs of transcription factors may instead be explained by the older duplication-degeneration-complementation model.

Whole genome re-sequencing reveals evolutionary patterns of sacred lotus (Nelumbo nucifera)

Sacred lotus(Nelumbo nucifera or lotus) is an important aquatic plant in horticulture and ecosystems. As a foundation for exploring genomic variation and evolution among different germplasms, we re-sequenced 19 individuals from three cultivated temperate lotus subgroups(rhizome,seed and flower lotus), one wild temperate lotus subgroup(wild lotus), one tropical lotus group(Thai lotus) and an outgroup(Nelumbo lutea). Through genetic diversity and polymorphism analysis by non-missing SNP sites widely distributed in the whole genome, we confirmed that wild and Thai lotus exhibited greater differentiation with a higher genomic diversity compared to cultivated lotus. Rhizome lotus had the lowest genomic diversity and a closer relationship to wild lotus, whereas the genomes of seed and flower lotus were admixed. Genes in energy metabolism process and plant immunity evolved rapidly in lotus, reflecting local adaptation.We established that candidate genes in genomic regions with significant differentiation associated with temperate and tropical lotus divergence always exhibited highly divergent expression pattern. Together, this study comprehensive and credible interpretates important patterns of genetic diversity and relationships, gene evolution, and genomic signature from ecotypic differentiation of sacred lotus.

Comparative analysis of whole genome structure of Streptococcus suis using whole genome PCR scanning

An outbreak associated with Streptococcus suis infection in humans emerged in Sichuan province, China in 2005. The outbreak is atypical for the apparent large number of human cases, high fatality rate and geographical spread. To determine whether the bacterium has changed, we compared both human and animal isolates from the Sichuan outbreak with those collected previously within China and in other countries using whole genome PCR scanning (WGPScaning) comparative sequencing of several known virulence factor genes and multilocus sequence typing (MLST) analysis. WGPScanning analysis showed that all primer pairs yielded PCR products of the expected sizes in all four strains tested. The nucleotide sequences of all the detected virulence factor genes are identical in the four strains and MLST results showed that the four isolates studied and reference strain all belonged to the ST1 com-plex. No new genetic changes were found in the genome structure of the isolates from this Sichuan outbreak.

Pitfalls of restriction enzyme analysis in identifying, characterizing,typing, and naming viral pathogens in the era of whole genome data, as illustrated by HAdV type 55

Restriction endonuclease analysis(REA),or restriction fragment length polymorphism(RFLP),was useful for identifying and determining the relatedness and putative identities of microbial strains(Tang et al.,1997)and for characterizing and discriminating large numbers of samples inexpensively in the past。

Lycophyte transcriptomes reveal two whole-genome duplications in Lycopodiaceae:Insights into the polyploidization of Phlegmariurus

Lycophytes are an ancient clade of the non-flowering vascular plants with chromosome numbers that vary from tens to hundreds.They are an excellent study system for examining whole-genome duplications(WGDs),or polyploidization,in spore-dispersed vascular plants.However,a lack of genome sequence data limits the reliable detection of very ancient WGDs,small-scale duplications(SSDs),and recent WGDs.Here,we integrated phylogenomic analysis and the distribution of synonymous substitutions per synonymous sites(Ks)of the transcriptomes of 13 species of lycophytes to identify,locate,and date multiple WGDs in the lycophyte family Lycopodiaceae.Additionally,we examined the genus Phlegmariurus for signs of genetic discordance,which can provide valuable insight into the underlying causes of such conflict(e.g.,hybridization,incomplete lineage sorting,or horizontal gene transfer).We found strong evidence that two WGD events occurred along the phylogenetic backbone of Lycopodiaceae,with one occurring in the common ancestor of extant Phlegmariurus(Lycopodiaceae)approximately 22-23 million years ago(Mya)and the other occurring in the common ancestor of Lycopodiaceae around 206-214 Mya.Interestingly,we found significant genetic discordance in the genus Phlegmariurus,indicating that the genus has a complex evolutionary history.This study provides molecular evidence for multiple WGDs in Lycopodiaceae and offers phylogenetic clues to the evolutionary history of Lycopodiaceae.

Efficient Haplotype Inference Algorithms in One Whole Genome Scan for Pedigree Data with Non-genotyped Founders

An efficient rule-based algorithm is presented for haplotype inference from general pedigree genotype data, with the assumption of no recombination. This algorithm generalizes previous algorithms to handle the cases where some pedigree founders are not genotyped, provided that for each nuclear family at least one parent is genotyped and each non-genotyped founder appears in exactly one nuclear family. The importance of this generalization lies in that such cases frequently happen in real data, because some founders may have passed away and their genotype data can no longer be collected. The algorithm runs in O（m^3n^3） time, where m is the number of single nucleotide polymorphism （SNP） loci under consideration and n is the number of genotyped members in the pedigree. This zero-recombination haplotyping algorithm is extended to a maximum parsimoniously haplotyping algorithm in one whole genome scan to minimize the total number of breakpoint sites, or equivalently, the number of maximal zero-recombination chromosomal regions. We show that such a whole genome scan haplotyping algorithm can be implemented in O（m^3n^3） time in a novel incremental fashion, here m denotes the total number of SNP loci along the chromosome.

Validation of the diagnostic potential of mtDNA copy number derived from whole genome sequencing

Diagnosis of mitochondrial DNA（mt DNA）disorders has traditionally been focused on the presence of point mutations and large deletions.However,deviations in mitochondrial abundance or mt DNA copy number can also be associated with many physiological and pathological conditions（Bai and Wong,2005）.

Enhanced osteogenesis of titanium with nano-Mg(OH)_(2) film and a mechanism study via whole genome expression analysis

Titanium(Ti)has been the most widely used orthopedic implant in the past decades.However,their inert surface often leads to insufficient osteointegration of Ti implant.To solve this issue,two bioactive Mg(OH)_(2) films were developed on Ti surfaces using hydrothermal treatment(Ti-M1#and Ti-M2#).The Mg(OH)_(2) films showed nano-flake structures:sheets on Ti-M1#with a thickness of 14.7±0.7 nm and a length of 131.5±2.9 nm,and on Ti-M2#with a thickness of 13.4±2.2 nm and a length of 56.9±5.6 nm.Both films worked as Mg ions releasing platforms.With the gradual degradation of Mg(OH)_(2) films,weakly alkaline microenvironments will be established surrounding the modified implants.Benefiting from the sustained release of Mg ions,nanostructures,and weakly alkaline microenvironments,the as-prepared nano-Mg(OH)_(2) coated Ti showed better in vitro and in vivo osteogenesis.Notably,Ti-M2#showed better osteogenesis than Ti-M1#,which can be ascribed to its smaller nanostructure.Moreover,whole genome expression analysis was applied to study the osteogenic mechanism of nano-Mg(OH)_(2) films.For both coated samples,most of the genes related to ECM-receptor interaction,focal adhesion,and TGF-β pathways were upregulated,indicating that these signaling pathways were activated,leading to better osteogenesis.Furthermore,cells cultured on Ti-M2#showed markedly upregulated BMP-4 gene expression,suggesting that the nanostructure with Mg ion release ability can better activate BMP-4 related signaling pathways,resulting in better osteogenesis.Nano-Mg(OH)_(2) films demonstrated a superior osteogenesis and are promising surface modification strategy for orthopedic applications.

Whole Genome Duplication of Intra- and Inter-chromosomes in the Tomato Genome

Whole genome duplication （WGD） events have been proven to occur in the evolutionary history of most angiosperms. Tomato is considered a model species of the Solanaceae family. In this study, we describe the details of the evolutionary process of the tomato genome by detecting collinearity blocks and dating the WGD events on the tree of life by combining two different methods： synonymous substitution rates （Ks） and phylogenetic trees. In total, 593 collinearity blocks were discovered out of 12 pseudo-chromosomes con- structed. It was evident that chromosome 2 had experienced an intra-chromosomat duplication event. Major inter-chromosomal dupli- cation occurred among all the pseudo-chromosome. We calculated the Ks value of these collinearity blocks. Two peaks of Ks distribution were found, corresponding to two WGD events occurring approximately 36-82 million years ago （MYA） and 148--205 MYA. Additionally, the results of phylogenetic trees suggested that the more recent WGD event may have occurred after the divergence of the rosidasterid clade, but before the major diversification in Solanaceae. The older WGD event was shown to have occurred before the divergence of the rosid-asterid clade and after the divergence of rice-Arabidopsis （monocot-dicot）.