Genome-wide Identification of the Class III POD Gene Family and their Expression Profiling in Grapevine (Vitisvinifera L)

Genome-wide Identification of the Class III POD Gene Family and their Expression Profiling in Grapevine (Vitisvinifera L) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Genome-wide Identification of the Class III POD Gene Family and their Expression Profiling in Grapevine (Vitisvinifera L) Huilin Xiao, Chaoping Wang, Nadeem Khan, Mengxia Chen, Weihong Fu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.16423/v2 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jun, 2020 Read the published version in BMC Genomics → Version 2 posted 9 You are reading this latest preprint version Show more versions Abstract Background : The class III peroxidases (PODs) are involved in a broad range of physiological activities, such as the formation of lignin, cell wall components, defense against pathogenicity or herbivore, and abiotic stress tolerance. The POD family members have been well-studied and characterized by bioinformatics analysis in several plant species, but no previous genome-wide analysis has been carried out of this gene family in grapevine to date. Results : We comprehensively identified 47 PODs in the grapevine genome and are further classified into 7 subgroups based on their phylogenetic analysis. Results of motif composition and gene structure organization analysis revealed that PODs in the same subgroup shared similar conjunction while the protein sequences were highly conserved. Intriguingly, the integrated analysis of chromosomal mapping and gene collinearity analysis proposed that both dispersed and tandem duplication events contributed to the expansion of PODs in grapevine. Also, the gene duplication analysis suggested that most of the genes (20) were dispersed followed by (15) tandem, (9) segmental or whole-genome duplication, and (3) proximal, respectively. The evolutionary analysis of PODs, such as Ka/Ks ratio of the 15 duplicated gene pairs were less than 1.00, indicated that most of the gene pairs exhibiting purifying selection and 7 pairs underwent positive selection with value greater than 1.00. The Gene Ontology Enrichment (GO), Kyoto Encyclopedia of Genes Genomics (KEGG) analysis, and cis-elements prediction also revealed the positive functions of PODs in plant growth and developmental activities, and response to stress stimuli. Further, based on the publically available RNA-sequence data, the expression patterns of PODs in tissue-specific response during several developmental stages revealed diverged expression patterns. Subsequently, 30 genes were selected for RT-PCR validation in response to (NaCl, drought, and ABA), which showed their critical role in grapevine. Conclusions : In conclusion, we predict that these results will lead to novel insights regarding genetic improvement of grapevine. Epigenetics & Genomics Grapevine Genome-wide analysis POD genes family Collinearity and Expression analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Peroxidases (EC 1.11.1.X) is a group of well-known large multi-gene family and that are broadly dispersed in living organisms. They catalyze oxidative reactions using hydrogen peroxide (H 2 O 2 ) as the electron acceptor in their active center with a metal. Based on their structure variations, the peroxidase (PODs) can be characterized into two main groups such as either heme PODs and non-heme PODs [ 1 ]. Meanwhile, the heme PODs can be ordered into two more sub-families like animal PODs and non-animal PODs [ 2 ]. The non-animal superfamily contains three major sub-distinct classes namely, class I, II, and III [ 3 ]. The class III peroxidases (EC 1.11.1.7) are abbreviated in various ways in previous studies (POX, POD, Px, PER, and Prx) and act as plant-specific oxidoreductases [ 3 , 4 ]. In this study, we will use the abbreviation for class III peroxidase as POD. The class III plant peroxidase (POD) plant is a plant-specific oxidoreductas, which is one of the many types of peroxidases that are widely distributed in animals, plants and microorganisms [ 3 ] . In plants growth, they are also known for their dual role in both cell wall hardening as well softening [ 5 ]. The PODs are involved in in various processes (e.g. lignification, plant defense, development, germination) and their mechanisms of action (substrate oxidation, regulation of reactive oxygen species and the formation of radicals), focusing specifically on lignification [ 6-10 ]. In recent times, due to the results of transcriptomic data, a large number of PODs have been accompanying numerous biological processes [ 11-13 ]. However, the direct role of this multi-gene family is still elusive and only a few studies have demonstrated their functional role [ 5 , 14-16 ]. For example, Arabidopsis thaliana and Populus trichocarpa PODs ( AtPrx72 and PtrPO21 ), play a significant role in lignification of the leaves [ 17 , 18 ]. The overexpression of the POD genes in A. thaliana ( AtPrx22, AtPrx39, and AtPrx69 ) improve cold tolerance [ 19 ]. Moreover, the cotton GhPOX1 has been studied for a higher production level of reactive oxygen species [ 20 ]. Several, POD genes in roots of Zea mays are known to regulate by methyl jasmonate, salicylic acid and pathogen elicitors [ 21 ]. Taken together, based on these results, the PODs play an important role in biological, physiological, and in response to stress stimuli, therefore, its comprehensive analysis is necessary to further explore its role in plant growth and development. In addition, the POD family members have been well-studied and characterized by bioinformatics analysis in several plant species including, 73 PODs in Arabidopsis thaliana [ 22 ], 138 in Oryza sativa [ 23 ], 93 in Populus trichocarpa [ 24 ], 102 in Medicago sativa [ 25 ], 119 in Zea mays [ 26 ] , 94 in Pyrus bretschneideri [ 27 ]. Nevertheless, to date, no previous genome-wide analysis has been carried out of this gene family in grapevine. While, a large number of genes in this family suggested their functional diversity among each individual proteins [ 12 ]. Grapevine ( Vitis vinifera L) is one of the widely popular and important fruit crops in the world [26]. A common goal of current plant genomics research is to create an expandable platform for global classification and analysis of plant gene family. Hence, it’s necessary to provide a foundation for future research. In the meantime, the availability of the grapevine genome (Version 2.1) facilitate the research in grapevine momentously for its genetic studies by improvement in the quality of berry. In the present study, we performed a wide-ranging bioinformatics analysis of POD gene family and verified their role against various stress responses (i-e., NaCl, drought, and ABA) in grapevine. In total, 47 genes were identified for the first time in the grapevine genome and were systematically analyzed by genome-wide approaches. Thus, the study including, physicochemical properties, phylogenetic relationships, chromosomal mapping, collinear correlation, gene duplication events, rate of substitution rates, motif composition and gene structure, promoter sequence analysis, GO and KEGG enrichment analysis, and expression profiling using RNA-seq data and RT-PCR analysis in response to salt, drought and Abscisic acid (ABA). In general, the results of our study will undoubtedly be helpful for future research on fruits crop species and pay the base for functional characterization of the PODs gene family. 2. Results 2.1 Characterization of POD Gene Family in Grapevine In this study, a total of 47 POD genes were identified from the grapevine genome and for simplicity, we denominated as VvPOD1-VvPOD47 based on their orthologous position with Arabidopsis thaliana . We also studied some useful information of PODs including, the protein identifier, chromosomal localization, coding sequence (CDS) length (bp), and various physicochemical properties such as, protein length (aa), molecular weight (MW) kDa, isoelectric point (PIs), and grand average of hydropathicity (GRAVY). While, the gene duplication types (i.e., dispersed, tandem, proximal, and segmental or whole-genome duplication) and subcellular localization analysis were also briefly studied for each of POD proteins (Supplementary Table S1). In brief, the CDS length varies from 801bp ( VvPOD35 ) to 2188bp ( VvPOD12 ) with an average of 1009.021 bp. Similarly, the protein length varies from 266 aa ( VvPOD35 ) to 705 aa ( VvPOD12 ) with an average of 335.34 aa, respectively. Also, the MW ranged from 28.50 kDa ( VvPOD35 ) to 76.17 kDa ( VvPOD12 ) with mean MW of 36.48 kDa, the PIs varies from 4.16 ( VvPOD47 ) to 9.56 ( VvPG13 ), respectively. The results of GRAVY ranged from -0.37 ( VvPOD10 ) to 0.03 ( VvPOD25 ). Intriguingly, the variability was observed in most of the genes for GRAVY, indicating mostly hydrophilic properties and only a few of them ( VvPOD46 , VvPOD43 , VvPOD31 , and VvPOD25 ) are hydrophobic in nature by showing positive values. Additionally, the gene duplication analysis intimated that most of the genes (20) were dispersed followed by tandem (15), segmental or whole-genome duplication (9), and proximal (3), respectively. 2.2 Phylogenetic Relationships, Gene Structure Organization of POD Gene Family in Grapevine To investigate the evolutionary relationships, we used the 47 POD gene grapevine and 73 Arabidopsis thaliana to construct a maximum likelihood approach tree by using MEGA 7.0. The phylogenetic tree reveals that PODs can be further subcategorized into 7 subgroups (Figure 1). The results exhibited that there is an uneven distribution of VvPOD genes compared with AtPODs . For instance, we observed that subgroup 7 contains the most number of genes (15 and 17) as compared to other subgroups in grapevine and Arabidopsis . The phylogenetic tree also revealed the relatively close genetic relationships with Arabidopsis . The 10 conserved motifs ranging from (motif 1-10) of VvPODs were explored by the MEME program. Markedly, motifs 1-4 were most common among the members of PODs, suggesting unique features among subgroups (Figure 2a). Also, the LOGOS for these motifs were obtained by MEME, the higher number (100) consensus sequences were observed in motif-2 while a less number (50) were recorded in motif 4, and motifs 8-10, respectively (Supplementary Figure S1). The gene structure organization was analyzed based on CDS and untranslated regions (UTRs) by using TBtools. The result reveals that VvPOD members are highly conserved within each other and displayed a similarity among subgroups (Figure 2b). Further, these findings indicated the structural diversification among VvPOD gene family. 2.3 Chromosomal Localization, Gene Collinearity, and Ka/Ks Analysis of POD To illustrate the chromosomal localization among 47 POD members and the gene collinearity analysis between grapevine and Arabidopsis were drawn with the help of TBtools software. The results for PODs chromosomal localization unveiled the irregular distribution patterns ranging from 1-9 proteins per chromosome except (chr5, chr9, and chr15) across 19 different chromosomes (i.e., Chr01-Chr19) in the grapevine genome. Also, the number of genes on each chromosome were distinct such as the high number of genes (9) were observed on Chr12, followed by Chr1 and Chr18 each with 5 genes, chr6 has 4 genes, while 3 genes were allocated on the Chr7, Chr10 and unknown chromosome (ChrUn), respectively, as described in Figure 3. Thus, among POD members high variation patterns were observed in the grapevine genome. Furthermore, the gene collinearity relationships between V. vinifera ( VvPOD ) and Arabidopsis ( AtPOD ) was also illustrated by using circos plot with the help of TBtools software. As a consequence, high conservation was observed between VvPOD and AtPOD genes (Figure 3). The selection pressure among various types of duplications (i.e., dispersed, tandem, proximal, segmental or WGD), also intended by calculating the rates of synonymous substitution ( Ks ) and non-synonymous substitution ( Ka ). During evolutionary processes, the genes are typically exposed to various types of selection pressure, such as purifying selection ( Ka/Ks1), and neutral selection ( Ka/Ks =1) [ 28 ]. Among 47 VvPOD members, we selected 22 pairs (i.e., 10 pair dispersed, 1 pair proximal, 7 pair tandem, and 4 pair segmental or WGD) as presented in Table 1. Results showed that most of the gene pairs having less than 1.00 Ka/Ks ratio suggested purifying selection, thus revealed limited divergence after gene duplications. Though, 7 pairs were observed with higher than 1.00 values, implicating positive selection. 2.4 Gene Ontology Enrichment (GO), Kyoto Encyclopedia of Genes Genomics (KEGG) and Cis-Regulatory Elements Analysis in Grapevine The GO enrichment analysis for POD genes was performed to understand their functional regulatory mechanism by using the orthologous pairs of Arabidopsis thaliana . The three common subgroups were observed such as molecular functions (MF), cellular component (CC), and biological process (BP). In the MF processes, “oxidoreductase and catalytic activity” (GO:0016491 and GO:0003824), are highly enriched GO terms. Similarly, for CC processes and BP most of the GO terms are responsive to “cell wall, plasmodesma, symplast, cell-cell junction, plant-type cell wall” (GO:0005618, GO:0009506, GO:0055044, GO:0005911, and GO:0009505), and “response to toxic substance, cellular response to stimulus, oxidation-reduction process, metabolic and cellular process” (GO:0009636, GO:0051716, GO:0055114, GO:0008152, and GO:0009987), and are briefly summarized in Supplementary Table S2. As results, the GO terms for MF, CC, and BP, suggested the crucial role of PODs in various activities of grapevine. Additionally, the KEGG enrichment analysis indicated the three major pathways among PODs in grapevine such as “Biosynthesis of other secondary metabolites, phenylpropanoid biosynthesis, and metabolism (Supplementary Table S3). Moreover, the cis-acting elements in the promoter region of POD members were performed by using the PlantCARE database. In brief, most of the genes were largely participating in light regulation with key regulatory elements (GT1-motif, G-Box, GATA-motif, and AE-Box), followed by hormones (CGTCA-motif, TGACG-motif, ABRE, and GARE-motif), stress and other regulatory factors (LTR, ARE, CCAAT-Box, CAT-BOX, o2-site,), and circadian, respectively. Thus, we observed the diversified role of POD members and their indirect involvement in several biotic-abiotic/hormone signaling processes (Supplementary Table S4). 2.5 Expression Profiling of POD Genes in Different Organs and Developmental Stages in Grapevine The expression profiling of PODs in grapevine was based on 19 various tissue and organs during their developmental stages, the RNA-seq data were retrieved from NCBI (GSE36128) according to the previously reported study [ 29 ]. To represent the spatio-temporal expression, a heatmap was generated on FPKM-based (Log 2 ) values of 47 VvPOD genes (Figure 4) and Supplementary Table S5. Results revealed that 9 genes ( VvPOD1, VvPOD2, VvPOD6, VvPOD10, VvPOD12, VvPOD27, VvPOD32, VvPOD37 , and VvPOD46 ) displayed a striking expression among all tissues and organs, implicating their major participation in tissue-specific response and development. About more than 15 genes, were highly expressed in root, suggesting their important role in tissue-specific. Moreover, the rest of the genes showed either moderate or weak expression abundance in all the selected tissues and organs, speculating their limited response in grapevine. 2.6 qRT-PCR Analysis of POD Genes in Response to (NaCl, drought, and ABA) To investigate the role of VvPOD genes under diverse abiotic stress conditions, we performed qRT-PCR analysis of randomly selected 30 candidate genes and that were subjected to NaCl, drought, and ABA stress treatment. The results directed that all the genes responded variably and showed higher, moderate or low expression level compared to the controls. In response to salt stress, approximately 52% of the total genes showed higher expression level, whereas the rest of the genes showed either moderate or low expression. Interestingly, in the case of ABA and drought stress, about 78% and 72% genes were observed to be down-regulated (Figure 5a). Most of the genes decreased their expression at the early stress periods (1h and 12 h), but they tended to increase their expression afterwards (24h). The expression of seven genes ( VvPOD8 , VvPOD12 , VvPOD19 , VvPOD24 , VvPOD29 , VvPOD38 , VvPOD39 , and VvPOD40 ) was increased 24 hours after the treatment under all the stress conditions at; whereas only the transcripts of VvPOD4034 and VvPOD37 were decreased. Moreover, the correlation analysis based on Pearson’s Correlation Coefficient (PCC) of the relative expression indicated largely a highly positive correlation and some of them were found with inverse correlation (Figure 5b). Taken together, these results of POD genes based on expression level respond to multiple stresses and might play an important role in the maintenance of plant growth. 3. Discussion The PODs multi-gene family are involved in the various biological process by regulating plant growth and developmental processes. While, POD family members have been comprehensively analyzed by genome-wide approaches in several species including, Arabidopsis thaliana [ 22 ], Oryza sativa [ 23 ], Panicum virgatum [ 30 ], Populus trichocarpa [ 24 ], Medicago sativa [ 25 ], Zea mays [ 26 ] , Pyrus bretschneideri [ 27 ]. However, to date, no previous bioinformatics analysis have been carried out in grapevine for this important gene family. Also, the available genomic resources for grapevine (http://genomes.cribi.unipd.it/grape/) provides useful information and tools for the analysis of POD gene family in grapevine. In this study, a total of 47 POD genes were identified in grapevine and is known to be the largest gene families in woody plants [ 27 ]. We comprehensively analyzed physicochemical properties, phylogenetic relationships, chromosomal mapping, gene collinearity analysis, motif composition and gene structure organization, and evolutionary analysis for the duplicated pairs of POD. GO, KEGG, cis-regulatory elements, expression profiling of spatio-temporal response, and qRT-PCR analysis in response to (NaCl, drought, and ABA) disclosed extensive information on the gene functions and expression dynamics of tissue-specific and abiotic stress response in grapevine. We determined the phylogenetic relationships between grapevine ( VvPOD ) and ( AtPOD ) by comparative analysis. Results revealed an identical domain composition of VvPOD with the model plant. The phylogenetic tree was categorized into 7 subgroups and the result of our tree are consistent with previously reported study of PODs in Cassava [ 31 ]. The motif composition analysis also demonstrated that motifs 1-4 are common among all the POD members with highly conserved nature. Moreover, the comparative structure analysis of POD showed that same subgroup shared a common junction. These results indicated a possible structural diversification within VvPOD gene family, which plays an important role during the evolution of multi-gene family [ 32 ]. Gene duplications are the vital force in the process of genomic evolution and functional divergence [ 33 ]. Importantly, the gene duplication is considered a major component in the establishment of new genetic functions and evolutionary novelty [ 34 , 35 ]. Similarly, in the process of evolutionary history, most of the higher plant underwent polyploidization that is vital ingredient in shaping plant genome [ 36 ]. In this study, the types of duplications in grapevine were identified by the help of MCScanX among POD genes. Results showed 4 types of duplications including, dispersed (20), tandem (15), segmental or whole-genome duplication (9), and proximal (3). It is noteworthy that during the process of evolution segmental and tandem duplication plays a critical role in the expansion of gene family [ 27 , 37 , 38 ]. Though, in this study, we determined that both dispersed and tandem duplications contributed mainly to the expansion of POD gene family in grapevine. The selection pressure analysis are mainly based on three selection such as purifying ( Ka/Ks 1), and neutral selection ( Ka/Ks =1), and its evolutionary assessment provide useful guidelines during the rate of divergence [ 39 ]. Results inferred that 15 pairs of POD have shown less than 1.00 Ka/Ks ratio, indicating purifying selection and 7 pairs exhibiting more than 1.00 suggested the positive selection. The results of our study are also in consistent with previous reported studies on PODs [ 37 , 40 ]. In this study, we utilized the publically available RNA-seq data of 19 different tissues and organs to validate the tissue-specific response of 47 VvPODs . Among them, 9 genes ( VvPOD1, VvPOD2, VvPOD6, VvPOD10, VvPOD12, VvPOD27, VvPOD32, VvPOD37, and VvPOD46 ) exhibited significantly higher expression level among all the tissue and organs. The rest of the other genes showed down-regulation with similar tendency throughout grapevine tissues and organs developmental phases. To better understand the gene functions, expression profiling mainly provides a valuable clue to its regulatory role against specific treatment. Abiotic stresses considerably harbor plant growth and productivity, thus lead to economic losses. The drought severity hampered grapevine productivity and has deleterious impacts on viticulture worldwide [ 41 , 42 ]. Salt stress is one of the severe abiotic stress, while grapevine plants generally modify their physiology to combat salt stress severity [ 43 ]. In regulating plant tolerance to several abiotic stress, an eminent hormone, such as ABA plays an essential role. Many researchers have indicated that plant peroxidases are involved in various cellular processes during plant growth and development, and its response to abiotic and biotic stress have been reported over the years [ 26 , 44 ]. For instance, membrane-bounded peroxidase ( pmPOX1 , pmPOX2a , pmPOX2b, and pmPOX3 ) in Zea mays roots are regulated by methyl jasmonate, salicylic acid and pathogen elicitors [ 21 ]. Similarly, the overexpression of CpPrx01 altered the growth pattern of plants and condensed the level of a well-known hormone, indole-3-acetic acid (IAA) [ 45 ]. Thus, in this study, the qRT-PCR validation of 30 POD genes in response to NaCl, drought, and ABA displayed a range of differential expression. The salt stress increased the expression of most POD s, while drought and ABA stress decreased their expression. This suggested their important roles in grapevine against abiotic stresses. To improve and comprehend the possible transcriptional regulation functions of PODs, we also analyzed the GO, KEGG enrichment, and cis-elements in grapevine. Thus, these results inferred their diversified functions and indirect involvement in several biological processes, such as biotic-abiotic/hormone signaling. 4. Conclusions In conclusion, we systematically identified a total of 47 POD genes in grapevine and were categorized into 7 subgroups, as supported by phylogenetic analysis. The GO, KEGG, and cis-elements analysis also extended our repositories on the diverse functions of PODs in plant developments during various stress-related activities in grapevine. While, the transcriptional profiling of various organs during several developmental stages and RT-PCR analysis, provide a stand-point of the PODs in improving the plant growth. Thus, the results of our study increase our understanding of POD genes in grapevine and laying the solid foundation for genetic improvement in other fruit crops. 5. Methods 5.1 Identification of POD Genes Family in Grapevine In order to identify the POD genes, we used the 73 reference sequences of Arabidopsis against grapevine (genome version 2.1) with the help of BioEdit tools. In general, we retrieved the sequences of grapevine and Arabidopsis from online sources such as ensembl (https://plants.ensembl.org/index.html) and TAIR ( http://www.arabidopsis.org/ ). The domain composition was verified by using the NCBI-Conserved Domain database ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) and SMART databases ( http://smart.embl-heidelberg.de/ ) [ 46 ]. Those sequences with absent of POD domains and sequence with obvious error in length (>100aa length) were eliminated from the study before analysis. 5.2 Phylogenetic Analysis of POD Gene Family For phylogenetic characterization, the multiple sequences alignment (MSA) of PODs was performed by MUSCLE [ 47 ] using MEGA 7.0 software with the default options [ 48 ]. The phylogenetic trees were constructed using the maximum likelihood (ML) method. After scrutiny of several models in MEGA 7.0, we choose the Jones, Taylor, and Thornton amino acid substitution model (JTT model) and for the reliability of resulting phylogenetic tree, the bootstrap values of 1000 replications were performed and keeping the other parameters as a default. 5.3 Calculation of Synonymous (Ks) and Non-synonymous (Ka) for Duplicated Genes The rate of Ka/Ks was carried out for various types of duplicated pairs (i.e., dispersed, proximal, tandem, and segmental) by using MEGA 7.0 [ 48 ]. The Ks and Ka ratio was intended by Nei- Gojobori method (Juke-Cantor) model in MEGA 7.0 with the bootstrap values of 1000 replicates. The various types of duplications of the POD gene pairs were detected by the MCScan algorithm. 5.4 Gene Structure, Motifs Composition, and Physicochemical Analysis of POD Protein For the gene structure illustration, we utilized the GFF3 file of the grapevine genome and images were implemented by TBtools software [ 49 ]. The motifs analysis of POD protein were performed by the Multiple Em of Motif Elicitation (MEME Suite) version 5.0.5 and then demonstrated by TBtools software. The following parameter was calibrated for this purpose as follows: the maximum number of motifs 10, with a minimum width of 50 and a maximum of 100 and the other parameter was set as default [ 50 ]. While, for each gene of POD, the several physicochemical properties (i.e., molecular weight (MW), isoelectronic points (PIs), and GRAVY) were intended by ExPASY PROTPARAM tools ( http://web.expasy.org/protparam/ ). The subcellular localization was further predicted by using the WOLF PSORT online server ( https://wolfpsort.hgc.jp/ ). 5. 5 Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomics (KEGG) and Cis-Elements Predictions of Class III Peroxidase Gene Family For the GO and KEGG enrichment analysis, the online panther server ( http://pantherdb.org/ ) and the genome server was implemented (https://www.genome.jp/kegg/pathway.html) and their enriched pathways were further explored by TBtools software [ 49 ]. The POD promoter sequences (i.e., selected as 1500bp) was initially imported in Generic File Format (GFF) from the grapevine genome. After that various cis-regulatory elements for each promoter sequence was identified by the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) [ 51 ]. 5.6 Chromosomal Mapping and Gene Collinearity Analysis of Grapevine and Arabidopsis The grapevine genomic database (CRIBI. Available online: http://genomes.cribi.unipd.it/grape/, V2.1), was utilized for the chromosomal locations of POD genes and were mapped based on information available. Similarly, for gene collinearity analysis, the grapevine and Arabidopsis relationship was verified and demonstrated with the help of Circos (TBtools software) program was applied [ 49 ]. 5.7 Plant Material and Methods In the present study, we used the two-year-old potted grapevine plants ( V. vinifera cv. Summer Black), selected from greenhouse condition (25 ± 5 °C) under 16-h light/8-h dark photoperiod and 65% relative humidity (RH) at the Nanjing Agricultural University Nanjing-China. The grapevine plants were subjected to abiotic stress including, NaCl (100 mM), drought (irrigated with 15% (w/v) polyethylene glycol), and ABA (100 μM/100 mL of water) with an interval of 1, 6, 24 h against control (CK), with three biological replicates and the three samples were mixed to make one composite sample while using the 4th unfolded leaf from cv. Summer Black grapevine. At last, all the samples were quickly frozen in liquid nitrogen and stored at −80 °C for further use. 5.8 RNA Isolation and Transcriptional Profiling of POD Gene Family in Grapevine Total RNA was extracted from the grapevine leaves with the help of Trizol (Invitrogen) according to the manufacturer’s instructions. Then, the Primer Script RT reagent kit (TAKARA, Dalian, China) was used to convert the RNA into reverse-transcribed into cDNA. The gene-specific primers (Supplementary Table S6) was designed by the help of Becan Designer 7.9 and their specificity was confirmed using the BLAST tool against the grapevine genome. Also, we performed the RT-PCR by following the previously reported studies [ 52 , 53 ], and the relative fold expression was calculated using the comparative Ct-method. The expression patterns of all POD gene were analyzed based on a previous study [ 38 , 54 ], and for reference gene in qRT- PCR, we used the housekeeping actin gene ( AB073011 ). In summary, the real-time PCR amplification reactions were performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA) using SYBR Green (Applied Biosystems, CA, USA) with three replicates. While the following PCR parameter conditions were set as follow: denaturation at 95 °C for 2 min, 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 40 s, and extension at 72 °C for 15 s. For expression profiling, we utilized the RNA-sequence data from the NCBI GEO website (https://www.ncbi.nlm.nih.gov/geo/) under the series entry GSE36128. Further, the expression levels were quantified by FPKM (fragments per kilobase of transcript per million fragments mapped), and heat maps were generated by using Rstudio (R program package) based on log 2 FPKM values. Abbreviations PODs: peroxidase ABA: Abscisic acid CSD: coding sequence MW: molecular weight PIs: isoelectric point GRAVY: grand average of hydropathicity WGD: the whole genome duplication GO: Gene Ontology Enrichment KEGG: Kyoto Encyclopedia of Genes Genomics MF: Molecular Functions CC: Cellular Component BP: biological process PCC: Pearson’s Correlation Coefficient FPKM: Fragments per kilobase of transcript per million fragments mapped Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and materials: All data generated or analysed during this study are included in this published article and its supplementary information files Competing interests: The authors declare that they have no competing interests. Funding: This study was financially supported by The National Key Research and Development Project (2018YFD1000200), The Key Research and Development Project of Jiangsu Province (BE2018389), Jiangsu Planned Projects for Postdoctoral Research Funds, National Natural Science Foundation of China (31801809, 31872047). Authors' contributions: H. X., C. W., L. G., X. L. conceived and designed the experiments, H. X., C. W. performed the experiment: N. K., M. C., W. F. analyzed the data: L.G., X. L., wrote the manuscript. All authors have read and approved the manuscript Acknowledgements: Not applicable References Welinder KG: Superfamily of plant, fungal and bacterial peroxidases . Current Opinion in Structural Biology 1992, 2 (3):388-393. Mathé C, Barre A, Jourda C, Dunand C: Evolution and expression of class III peroxidases . Archives of Biochemistry and Biophysics 2010, 500 (1):58-65. Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H: A large family of Class III plant peroxidases . 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Nucleic acids research 2009, 37 (Web Server issue):W202-W208. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S: PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences . Nucleic Acids Research 2002, 30 (1):325-327. Khan N, Hu C-m, Amjad Khan W, Naseri E, Ke H, Huijie D, Hou X: Evolution and expression divergence of E2 gene family under multiple abiotic and phytohormones stresses in Brassica rapa . BioMed Research International 2018, 2018 :18. Khan N, Hu C-m, Khan WA, Wang W, Ke H, Huijie D, Zhishuo Z, Hou X: Genome-wide identification, classification, and expression pattern of homeobox gene family in Brassica rapa under various stresses . Scientific Reports 2018, 8 (1):16265. Shangguan L, Fang X, Chen L, Cui L, Fang J: Genome-wide analysis of autophagy-related genes (ARGs) in grapevine and plant tolerance to copper stress . Planta 2018, 247 (6):1449-1463. Tables Table.1. The POD genes in grapevine with outlier Ka/Ks and various types of duplications of the POD gene pairs with the detection by the MCScan algorithm (i.e., Dispersed, proximal, tandem, and segmental). Gene 1 Gene 2 Ks Ka Ka / Ks Selection Pressure Gene Duplications VvPOD1 VvPOD2 0.76 0.65 0.86 Purifying Selection Dispersed VvPOD3 VvPOD5 0.69 0.58 0.83 Purifying Selection Dispersed VvPOD9 VvPOD10 0.68 0.63 0.92 Purifying Selection Dispersed VvPOD11 VvPOD14 0.89 0.65 0.73 Purifying Selection Dispersed VvPOD15 VvPOD16 0.04 0.01 0.28 Purifying Selection Dispersed VvPOD17 VvPOD18 1.41 0.11 0.08 Purifying Selection Dispersed VvPOD25 VvPOD26 0.92 0.62 0.68 Purifying Selection Dispersed VvPOD27 VvPOD30 0.56 0.74 1.33 Positive Selection Dispersed VvPOD31 VvPOD32 0.60 0.64 1.07 Positive Selection Dispersed VvPOD42 VvPOD43 0.95 0.36 0.38 Purifying Selection Dispersed VvPOD24 VvPOD35 0.75 0.43 0.58 Purifying Selection Proximal VvPOD4 VvPOD12 1.20 0.45 0.38 Purifying Selection Tandem VvPOD13 VvPOD19 0.50 0.53 1.05 Positive Selection Tandem VvPOD20 VvPOD21 0.18 0.04 0.24 Purifying Selection Tandem VvPOD22 VvPOD23 0.24 0.56 2.35 Positive Selection Tandem VvPOD33 VvPOD34 0.40 0.05 0.13 Purifying Selection Tandem VvPOD38 VvPOD36 0.20 0.09 0.47 Purifying Selection Tandem VvPOD40 VvPOD41 0.07 0.03 0.35 Purifying Selection Tandem VvPOD6 VvPOD7 0.22 0.37 1.68 Positive Selection WGD or Segmental VvPOD8 VvPOD28 0.57 0.67 1.19 Positive Selection WGD or Segmental VvPOD29 VvPOD36 0.48 0.61 1.28 Positive Selection WGD or Segmental VvPOD37 VvPOD45 0.66 0.63 0.96 Purifying Selection WGD or Segmental Supplementary Legends Supplementary Figure: Figure S1.The LOGOS of PODS were also elucidated by MEME online server. Supplementary Tables: Table S1. The basic information of PODsgenes identified in grapevine. Table S2. The gene ontology (GO) of POD genes in grapevine. Table S3. Kyoto Encyclopedia of Genes and Genomics (KEGG) of POD genes in grapevine. Table S4. Cis-Elements of PODgenes in grapevine. Table S5. Relative expression patterns (log2) of POD genes in grapevine by qRT-PCR. Table S5S6.Sequences of the PODgenes primers used for quantitative real-time PCR. Supplementary Files SupplementaryFigure1.pdf SupplementaryTables.xlsx Cite Share Download PDF Status: Published Journal Publication published 29 Jun, 2020 Read the published version in BMC Genomics → Version 2 posted Editorial decision: Major revision 24 Apr, 2020 Review # 1 received at journal 23 Apr, 2020 Reviewer # 2 agreed at journal 01 Apr, 2020 Review # 2 received at journal 01 Apr, 2020 Reviewers invited by journal 31 Mar, 2020 Reviewer # 1 agreed at journal 31 Mar, 2020 Editor assigned by journal 30 Mar, 2020 Submission checks completed at journal 29 Mar, 2020 Editor invited by journal 29 Mar, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7066","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":455206,"identity":"57b3f1ae-f0b9-43ae-8540-74182289cb3f","order_by":1,"name":"Huilin Xiao","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huilin","middleName":"","lastName":"Xiao","suffix":""},{"id":455207,"identity":"8ec4df38-5523-489b-b69a-7a3f70cf09a6","order_by":2,"name":"Chaoping Wang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chaoping","middleName":"","lastName":"Wang","suffix":""},{"id":455208,"identity":"afce7049-830e-4bf8-a555-34de9170224d","order_by":3,"name":"Nadeem Khan","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Nadeem","middleName":"","lastName":"Khan","suffix":""},{"id":455209,"identity":"ed179bfa-50e3-466f-8d87-0e1239f2ac2b","order_by":4,"name":"Mengxia Chen","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mengxia","middleName":"","lastName":"Chen","suffix":""},{"id":455210,"identity":"583b7d9b-dd0d-4ef9-8b03-06976b1b191c","order_by":5,"name":"Weihong Fu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Weihong","middleName":"","lastName":"Fu","suffix":""},{"id":455211,"identity":"96cebdff-abb3-453b-9e70-5ed0c784ffae","order_by":6,"name":"Le Guan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYLCCBww2DIwNIBYbsVoSGNJI13IYyiJGi3xE8rMHCRXn85jbzxgwfCg7zMA/uwG/FsMbaeYGCWduFzP25Bgwzjh3mEHizgECWmYkmEkktt1ObGzIMWDmbTvMYCCRQEhL+jeJxH/nEhv73xgw/yVGi7xEDtCWhgOJjTOAtjASo8WA502ZRMKxZKCWZwUHe86l80jcIGRLe/o2iQ81dokb+5M3PvhRZi3HP4OQLQdgnmpgYACxefCrB9nSAGMQVDoKRsEoGAUjFgAA2lBFvYi3LlUAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Le","middleName":"","lastName":"Guan","suffix":""},{"id":455212,"identity":"f583a52b-0229-4ef0-8e19-5ad558fc771b","order_by":7,"name":"Xiangpeng Leng","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiangpeng","middleName":"","lastName":"Leng","suffix":""}],"badges":[],"createdAt":"2019-10-19 13:50:13","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.2.16423/v2","doiUrl":"https://doi.org/10.21203/rs.2.16423/v2","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-020-06828-z","type":"published","date":"2020-06-29T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":823758,"identity":"ac4c462f-1cb7-4f3c-a9a1-33f1c5546af2","added_by":"auto","created_at":"2020-04-02 14:59:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":137211,"visible":true,"origin":"","legend":"Phylogenetic relationship of POD genes between grapevine and Arabidopsis. The phylogenetic tree was constructed by MEGA 7.0 using the Maximum Likelihood Method (1000 bootstrap).","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/1.png"},{"id":823760,"identity":"5b89ebfb-d687-47ba-b01b-bfaf86eea8cc","added_by":"auto","created_at":"2020-04-02 14:59:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94717,"visible":true,"origin":"","legend":"(A) Motif composition of POD in grapevine are presented in different color ranging from motif 1-10. (B) The coding sequences (CDS) and untranslated regions (UTR) for PODs in grapevine are represented by yellow and green boxes, respectively. Motif composition and gene structure were visualized by TBtools software. At the bottom of the figure, the relative position is proportionally displayed based on the kilobase scale.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/2.png"},{"id":823762,"identity":"51f842d1-d683-4ec4-ac82-29a57e7250be","added_by":"auto","created_at":"2020-04-02 14:59:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131579,"visible":true,"origin":"","legend":"(A) The chromosomal localizations are shown for grapevine (Chr01-19) is blue and for Arabidopsis different random colors. (B) The collinear correlation at the center for all the POD genes is displayed between grapevines and Arabidopsis. The green line indicates the collinear relationship among VvPODs and AtPODs, blue represent the relation within VvPOD and red lines indicates the tandem duplications.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/3.png"},{"id":823763,"identity":"bdc1cfd4-ff29-4f5a-8d03-584c3ad54130","added_by":"auto","created_at":"2020-04-02 14:59:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":240970,"visible":true,"origin":"","legend":"Expression profiles of the POD genes in different grapevine organs, tissues, and developmental stages. Data were normalized based on the mean expression value of each gene in all tissues analyzed. BerryPericarp-FS: berry pericarp fruit set; BerryPericarp-PFS: berry pericarp post-fruit set; BerryPericarp-V: Bud-S: bud swell; Bud-B: bud burst (green tip); Bud-AB: bud after-burst (rosette of leaf tips visible); Bud-L: latent bud; Bud-W: winter bud; Flower-FB: flowering begins (10% caps off); Flower-F: flowering (50% caps off); Leaf-Y: young leaf (pool of leaves from shoot of 5 leaves); Leaf-FS: mature leaf (pool of leaves from shoot at fruit set); Rachis-FS: rachis fruit set; Rachis-PFS: rachis post fruit set; Stem-G: green stem; Stem-W: woody stem; Tendril-Y: young tendril (pool of tendrils from shoot of 7 leaves); Tendril-WD: well developed tendril (pool of tendrils from shoot of 12 leaves); Tendril-FS: mature tendril (pool of tendrils at fruit set).","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/4.png"},{"id":823764,"identity":"d8457cd5-8992-40cc-a945-844d777afb1e","added_by":"auto","created_at":"2020-04-02 14:59:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":239832,"visible":true,"origin":"","legend":"(A) Relative expression profiling by RT-PCR for significantly up and down-regulated genes under various abiotic stress (i.e., NaCl, PEG, and ABA). (B) Pearson's correlation coefficients (PCCs) of 30 VvPOD genes against abiotic stress such as NaCl, PEG, and ABA and were illustrated by using RStudio.","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/5.png"},{"id":13496703,"identity":"985252a6-aee5-46bb-b16f-15a8949ce78a","added_by":"auto","created_at":"2021-09-16 22:50:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2258440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/60ab60ae-04e4-45da-b559-ad99ac8930aa.pdf"},{"id":823761,"identity":"54a6ced2-8a7e-45b4-84d5-e621f8d50640","added_by":"auto","created_at":"2020-04-02 14:59:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":724955,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/Supplementary Figure 1.pdf"},{"id":823759,"identity":"bf57d7d6-8e97-4226-8cf0-8fbeca3aebd3","added_by":"auto","created_at":"2020-04-02 14:59:36","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":57932,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7066/v2/Supplementary Tables.xlsx"}],"financialInterests":"","formattedTitle":"Genome-wide Identification of the Class III POD Gene Family and their Expression Profiling in Grapevine (Vitisvinifera L)","fulltext":[{"header":"1. Background","content":"\u003cp\u003ePeroxidases (EC 1.11.1.X) is a group of well-known large multi-gene family and that are broadly dispersed in living organisms. They catalyze oxidative reactions using hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) as the electron acceptor in their active center with a metal. Based on their structure variations, the peroxidase (PODs) can be characterized into two main groups such as either heme PODs and non-heme PODs [\u003ca href=\"#_ENREF_1\"\u003e1\u003c/a\u003e]. Meanwhile, the heme PODs can be ordered into two more sub-families like animal PODs and non-animal PODs [\u003ca href=\"#_ENREF_2\"\u003e2\u003c/a\u003e]. The non-animal superfamily contains three major sub-distinct classes namely, class I, II, and III [\u003ca href=\"#_ENREF_3\"\u003e3\u003c/a\u003e]. The class III peroxidases (EC 1.11.1.7) are abbreviated in various ways in previous studies (POX, POD, Px, PER, and Prx) and act as plant-specific oxidoreductases [\u003ca href=\"#_ENREF_3\"\u003e3\u003c/a\u003e, \u003ca href=\"#_ENREF_4\"\u003e4\u003c/a\u003e]. In this study, we will use the abbreviation for class III peroxidase as POD. The class III plant peroxidase (POD) plant is a plant-specific oxidoreductas, which is one of the many types of peroxidases that are widely distributed in animals, plants and microorganisms [\u003ca href=\"#_ENREF_3\"\u003e3\u003c/a\u003e] . In plants growth, they are also known for their dual role in both cell wall hardening as well softening [\u003ca href=\"#_ENREF_5\"\u003e5\u003c/a\u003e]. The PODs are involved in in various processes (e.g. lignification, plant defense, development, germination) and their mechanisms of action (substrate oxidation, regulation of reactive oxygen species and the formation of radicals), focusing specifically on lignification [\u003ca href=\"#_ENREF_6\"\u003e6-10\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eIn recent times, due to the results of transcriptomic data, a large number of PODs have been accompanying numerous biological processes [\u003ca href=\"#_ENREF_11\"\u003e11-13\u003c/a\u003e]. However, the direct role of this multi-gene family is still elusive and only a few studies have demonstrated their functional role [\u003ca href=\"#_ENREF_5\"\u003e5\u003c/a\u003e, \u003ca href=\"#_ENREF_14\"\u003e14-16\u003c/a\u003e]. For example, \u003cem\u003eArabidopsis thaliana \u003c/em\u003eand \u003cem\u003ePopulus trichocarpa\u003c/em\u003e PODs (\u003cem\u003eAtPrx72 \u003c/em\u003eand \u003cem\u003ePtrPO21\u003c/em\u003e), play a significant role in lignification of the leaves [\u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e, \u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e]. The overexpression of the \u003cem\u003ePOD\u003c/em\u003e genes in \u003cem\u003eA. thaliana\u003c/em\u003e (\u003cem\u003eAtPrx22, AtPrx39, and AtPrx69\u003c/em\u003e) improve cold tolerance [\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e]. Moreover, the cotton \u003cem\u003eGhPOX1\u003c/em\u003e has been studied for a higher production level of reactive oxygen species [\u003ca href=\"#_ENREF_20\"\u003e20\u003c/a\u003e]. Several, \u003cem\u003ePOD\u003c/em\u003e genes in roots of \u003cem\u003eZea mays\u003c/em\u003e are known to regulate by methyl jasmonate, salicylic acid and pathogen elicitors [\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e]. Taken together, based on these results, the PODs play an important role in biological, physiological, and in response to stress stimuli, therefore, its comprehensive analysis is necessary to further explore its role in plant growth and development.\u003c/p\u003e\n\u003cp\u003eIn addition, the POD family members have been well-studied and characterized by bioinformatics analysis in several plant species including, 73 PODs in \u003cem\u003eArabidopsis thaliana \u003c/em\u003e[\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e], 138 \u0026nbsp;in \u003cem\u003eOryza sativa \u003c/em\u003e[\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e], 93 in \u003cem\u003ePopulus trichocarpa \u003c/em\u003e[\u003ca href=\"#_ENREF_24\"\u003e24\u003c/a\u003e], 102 in \u003cem\u003eMedicago sativa \u003c/em\u003e[\u003ca href=\"#_ENREF_25\"\u003e25\u003c/a\u003e], 119 in \u003cem\u003eZea mays \u003c/em\u003e[\u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e]\u003cem\u003e, \u003c/em\u003e94 in \u003cem\u003ePyrus bretschneideri \u003c/em\u003e[\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e]. Nevertheless, to date, no previous genome-wide analysis has been carried out of this gene family in grapevine. While, a large number of genes in this family suggested their functional diversity among each individual proteins [\u003ca href=\"#_ENREF_12\"\u003e12\u003c/a\u003e]. Grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e L) is one of the widely popular and important fruit crops in the world [26]. A common goal of current plant genomics research is to create an expandable platform for global classification and analysis of plant gene family. Hence, it\u0026rsquo;s necessary to provide a foundation for future research. In the meantime, the availability of the grapevine genome (Version 2.1) facilitate the research in grapevine momentously for its genetic studies by improvement in the quality of berry.\u003c/p\u003e\n\u003cp\u003eIn the present study, we performed a wide-ranging bioinformatics analysis of \u003cem\u003ePOD\u003c/em\u003e gene family and verified their role against various stress responses (i-e., NaCl, drought, and ABA) in grapevine. In total, 47 genes were identified for the first time \u0026nbsp;in the grapevine genome and were systematically analyzed by genome-wide approaches. Thus, the study including, physicochemical properties, phylogenetic relationships, chromosomal mapping, collinear correlation, gene duplication events, rate of substitution rates, motif composition and gene structure, promoter sequence analysis, GO and KEGG enrichment analysis, and expression profiling using RNA-seq data and RT-PCR analysis in response to salt, drought and Abscisic acid (ABA). In general, the results of our study will undoubtedly be helpful for future research on fruits crop species and pay the base for functional characterization of the \u003cem\u003ePODs\u003c/em\u003e gene family.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cem\u003e2.1\u003c/em\u003e \u003cem\u003eCharacterization of POD Gene Family in Grapevine\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, a total of 47 \u003cem\u003ePOD\u003c/em\u003e genes were identified from the grapevine genome and for simplicity, we denominated as \u003cem\u003eVvPOD1-VvPOD47\u003c/em\u003e based on their orthologous position with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. We also studied some useful information of PODs including, the protein identifier, chromosomal localization, coding sequence (CDS) length (bp), and various physicochemical properties such as, protein length (aa), molecular weight (MW) kDa, isoelectric point (PIs), and grand average of hydropathicity (GRAVY). While, the gene duplication types (i.e., dispersed, tandem, proximal, and segmental or whole-genome duplication) and subcellular localization analysis were also briefly studied for each of POD proteins (Supplementary Table S1). In brief, the CDS length varies from 801bp (\u003cem\u003eVvPOD35\u003c/em\u003e) to 2188bp (\u003cem\u003eVvPOD12\u003c/em\u003e) with an average of 1009.021 bp. Similarly, the protein length varies from 266 aa (\u003cem\u003eVvPOD35\u003c/em\u003e) to 705 aa (\u003cem\u003eVvPOD12\u003c/em\u003e) with an average of 335.34 aa, respectively. Also, the MW ranged from 28.50 kDa (\u003cem\u003eVvPOD35\u003c/em\u003e) to 76.17 kDa (\u003cem\u003eVvPOD12\u003c/em\u003e) with mean MW of 36.48 kDa, the PIs varies from 4.16 (\u003cem\u003eVvPOD47\u003c/em\u003e) to 9.56 (\u003cem\u003eVvPG13\u003c/em\u003e), respectively. The results of GRAVY ranged from -0.37 (\u003cem\u003eVvPOD10\u003c/em\u003e) to 0.03 (\u003cem\u003eVvPOD25\u003c/em\u003e). Intriguingly, the variability was observed in most of the genes for GRAVY, indicating mostly hydrophilic properties and only a few of them (\u003cem\u003eVvPOD46\u003c/em\u003e, \u003cem\u003eVvPOD43\u003c/em\u003e, \u003cem\u003eVvPOD31\u003c/em\u003e, and \u003cem\u003eVvPOD25\u003c/em\u003e) are hydrophobic in nature by showing positive values. Additionally, the gene duplication analysis intimated that most of the genes (20) were dispersed followed by tandem (15), segmental or whole-genome duplication (9), and proximal (3), respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2\u003c/em\u003e \u003cem\u003ePhylogenetic Relationships, Gene Structure Organization of POD Gene Family in Grapevine\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the evolutionary relationships, we used the 47 \u003cem\u003ePOD\u003c/em\u003e gene grapevine and 73 \u003cem\u003eArabidopsis thaliana \u003c/em\u003eto construct a maximum likelihood approach tree by using MEGA 7.0. The phylogenetic tree reveals that PODs can be further subcategorized into 7 subgroups (Figure 1). The results exhibited that there is an uneven distribution of \u003cem\u003eVvPOD\u003c/em\u003e genes compared with \u003cem\u003eAtPODs\u003c/em\u003e. For instance, we observed that subgroup 7 contains the most number of genes (15 and 17) as compared to other subgroups in grapevine and \u003cem\u003eArabidopsis\u003c/em\u003e. The phylogenetic tree also revealed the relatively close genetic relationships with \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe 10 conserved motifs ranging from (motif 1-10) of VvPODs were explored by the MEME program. Markedly, motifs 1-4 were most common among the members of PODs, suggesting unique features among subgroups (Figure 2a). Also, the LOGOS for these motifs were obtained by MEME, the higher number (100) consensus sequences were observed in motif-2 while a less number (50) were recorded in motif 4, and motifs 8-10, respectively (Supplementary Figure S1). The gene structure organization was analyzed based on CDS and untranslated regions (UTRs) by using TBtools. The result reveals that \u003cem\u003eVvPOD\u003c/em\u003e members are highly conserved within each other and displayed a similarity among subgroups (Figure 2b). Further, these findings indicated the structural diversification among \u003cem\u003eVvPOD\u003c/em\u003e gene family.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3\u003c/em\u003e \u003cem\u003eChromosomal Localization, Gene Collinearity, and Ka/Ks Analysis of POD\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo illustrate the chromosomal localization among 47 POD members and the gene collinearity analysis between grapevine and \u003cem\u003eArabidopsis\u003c/em\u003e were drawn with the help of TBtools software. The results for PODs chromosomal localization unveiled the irregular distribution patterns ranging from 1-9 proteins per chromosome except (chr5, chr9, and chr15) across 19 different chromosomes (i.e., Chr01-Chr19) in the grapevine genome. Also, the number of genes on each chromosome were distinct such as the high number of genes (9) were observed on Chr12, followed by Chr1 and Chr18 each with 5 genes, chr6 has 4 genes, while 3 genes were allocated on the Chr7, Chr10 and unknown chromosome (ChrUn), respectively, as described in Figure 3. Thus, among POD members high variation patterns were observed in the grapevine genome. Furthermore, the gene collinearity relationships between \u003cem\u003eV. vinifera\u003c/em\u003e (\u003cem\u003eVvPOD\u003c/em\u003e) and \u003cem\u003eArabidopsis \u003c/em\u003e(\u003cem\u003eAtPOD\u003c/em\u003e) was also illustrated by using circos plot with the help of TBtools software. As a consequence, high conservation was observed between \u003cem\u003eVvPOD\u003c/em\u003e and \u003cem\u003eAtPOD\u003c/em\u003e genes (Figure 3).\u003c/p\u003e\n\u003cp\u003eThe selection pressure among various types of duplications (i.e., dispersed, tandem, proximal, segmental or WGD), also intended by calculating the rates of synonymous substitution (\u003cem\u003eKs\u003c/em\u003e) and non-synonymous substitution (\u003cem\u003eKa\u003c/em\u003e). During evolutionary processes, the genes are typically exposed to various types of selection pressure, such as purifying selection (\u003cem\u003eKa/Ks\u0026lt;\u003c/em\u003e1), positive selection (\u003cem\u003eKa/Ks\u003c/em\u003e\u0026gt;1), and neutral selection (\u003cem\u003eKa/Ks\u003c/em\u003e=1) [\u003ca href=\"#_ENREF_28\"\u003e28\u003c/a\u003e]. Among 47 \u003cem\u003eVvPOD\u003c/em\u003e members, we selected 22 pairs (i.e., 10 pair dispersed, 1 pair proximal, 7 pair tandem, and 4 pair segmental or WGD) as presented in Table 1. Results showed that most of the gene pairs having less than 1.00 \u003cem\u003eKa/Ks \u003c/em\u003eratio suggested purifying selection, thus revealed limited divergence after gene duplications. Though, 7 pairs were observed with higher than 1.00 values, implicating positive selection.\u0026nbsp;\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4\u003c/em\u003e \u003cem\u003eGene Ontology Enrichment (GO), Kyoto Encyclopedia of Genes Genomics (KEGG) and Cis-Regulatory Elements Analysis in Grapevine\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe GO enrichment analysis for \u003cem\u003ePOD\u003c/em\u003e genes was performed to understand their functional regulatory mechanism by using the orthologous pairs of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The three common subgroups were observed such as molecular functions (MF), cellular component (CC), and biological process (BP). In the MF processes, \u0026ldquo;oxidoreductase and catalytic activity\u0026rdquo; (GO:0016491 and GO:0003824), are highly enriched GO terms. Similarly, for CC processes and BP most of the GO terms are responsive to \u0026ldquo;cell wall, plasmodesma, symplast, cell-cell junction, plant-type cell wall\u0026rdquo; (GO:0005618, GO:0009506, GO:0055044, GO:0005911, and GO:0009505), and \u0026ldquo;response to toxic substance, cellular response to stimulus, oxidation-reduction process, metabolic and cellular process\u0026rdquo; (GO:0009636, GO:0051716, GO:0055114, GO:0008152, and GO:0009987), and are briefly summarized in Supplementary Table S2. As results, the GO terms for MF, CC, and BP, suggested the crucial role of PODs in various activities of grapevine.\u003c/p\u003e\n\u003cp\u003eAdditionally, the KEGG enrichment analysis indicated the three major pathways among PODs in grapevine such as \u0026ldquo;Biosynthesis of other secondary metabolites, phenylpropanoid biosynthesis, and metabolism (Supplementary Table S3).\u003cem\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMoreover, the cis-acting elements in the promoter region of POD members were performed by using the PlantCARE database. In brief, most of the genes were largely participating in light regulation with key regulatory elements (GT1-motif, G-Box, GATA-motif, and AE-Box), followed by hormones (CGTCA-motif, TGACG-motif, ABRE, and GARE-motif), stress and other regulatory factors (LTR, ARE, CCAAT-Box, CAT-BOX, o2-site,), and circadian, respectively. Thus, we observed the diversified role of POD members and their indirect involvement in several biotic-abiotic/hormone signaling processes (Supplementary Table S4).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5\u003c/em\u003e \u003cem\u003eExpression Profiling of POD Genes in Different Organs and Developmental Stages in Grapevine \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe expression profiling of PODs in grapevine was based on 19 various tissue and organs during their developmental stages, the RNA-seq data were retrieved from NCBI (GSE36128) according to the previously reported study [\u003ca href=\"#_ENREF_29\"\u003e29\u003c/a\u003e]. To represent the spatio-temporal expression, a heatmap was generated on FPKM-based (Log\u003csub\u003e2\u003c/sub\u003e) values of 47 \u003cem\u003eVvPOD\u003c/em\u003e genes (Figure 4) and Supplementary Table S5. Results revealed that 9 genes (\u003cem\u003eVvPOD1, VvPOD2, VvPOD6, VvPOD10, VvPOD12, VvPOD27, VvPOD32, VvPOD37\u003c/em\u003e, and \u003cem\u003eVvPOD46\u003c/em\u003e) displayed a striking expression among all tissues and organs, implicating their major participation in tissue-specific response and development. About more than 15 genes, were highly expressed in root, suggesting their important role in tissue-specific. Moreover, the rest of the genes showed either moderate or weak expression abundance in all the selected tissues and organs, speculating their limited response in grapevine.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.6\u003c/em\u003e \u003cem\u003eqRT-PCR Analysis of POD Genes in Response to (NaCl, drought, and ABA)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of \u003cem\u003eVvPOD\u003c/em\u003e genes under diverse abiotic stress conditions, we performed qRT-PCR analysis of randomly selected 30 candidate genes and that were subjected to NaCl, drought, and ABA stress treatment. The results directed that all the genes responded variably and showed higher, moderate or low expression level compared to the controls. In response to salt stress, approximately 52% of the total genes showed higher expression level, whereas the rest of the genes showed either moderate or low expression. Interestingly, in the case of ABA and drought stress, about 78% and 72% genes were observed to be down-regulated (Figure 5a). Most of the genes decreased their expression at the early stress periods (1h and 12 h), but they tended to increase their expression afterwards (24h). The expression of seven genes (\u003cem\u003eVvPOD8\u003c/em\u003e, \u003cem\u003eVvPOD12\u003c/em\u003e, \u003cem\u003eVvPOD19\u003c/em\u003e, \u003cem\u003eVvPOD24\u003c/em\u003e, \u003cem\u003eVvPOD29\u003c/em\u003e, \u003cem\u003eVvPOD38\u003c/em\u003e, \u003cem\u003eVvPOD39\u003c/em\u003e, and\u003cem\u003e VvPOD40\u003c/em\u003e) was increased 24 hours after the treatment under all the stress conditions at; whereas only the transcripts of \u003cem\u003eVvPOD4034\u003c/em\u003e and \u003cem\u003eVvPOD37\u003c/em\u003e were decreased. Moreover, the correlation analysis based on Pearson\u0026rsquo;s Correlation Coefficient (PCC) of the relative expression indicated largely a highly positive correlation and some of them were found with inverse correlation (Figure 5b). Taken together, these results of \u003cem\u003ePOD\u003c/em\u003e genes based on expression level respond to multiple stresses and might play an important role in the maintenance of plant growth.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe PODs multi-gene family are involved in the various biological process by regulating plant growth and developmental processes. While, POD family members have been comprehensively analyzed by genome-wide approaches in several species including, \u003cem\u003eArabidopsis thaliana \u003c/em\u003e[\u003ca href=\"#_ENREF_22\"\u003e22\u003c/a\u003e], \u003cem\u003eOryza sativa \u003c/em\u003e[\u003ca href=\"#_ENREF_23\"\u003e23\u003c/a\u003e], \u003cem\u003ePanicum virgatum\u003c/em\u003e [\u003ca href=\"#_ENREF_30\"\u003e30\u003c/a\u003e], \u003cem\u003ePopulus trichocarpa \u003c/em\u003e[\u003ca href=\"#_ENREF_24\"\u003e24\u003c/a\u003e], \u003cem\u003eMedicago sativa \u003c/em\u003e[\u003ca href=\"#_ENREF_25\"\u003e25\u003c/a\u003e], \u003cem\u003eZea mays \u003c/em\u003e[\u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e]\u003cem\u003e,\u003c/em\u003e \u003cem\u003ePyrus bretschneideri \u003c/em\u003e[\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e]. However, to date, no previous bioinformatics analysis have been carried out in grapevine for this important gene family. Also, the available genomic resources for grapevine (http://genomes.cribi.unipd.it/grape/) provides useful information and tools for the analysis of \u003cem\u003ePOD\u003c/em\u003e gene family in grapevine. In this study, a total of 47 \u003cem\u003ePOD\u003c/em\u003e genes were identified in grapevine and is known to be the largest gene families in woody plants [\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e]. We comprehensively analyzed physicochemical properties, phylogenetic relationships, chromosomal mapping, gene collinearity analysis, motif composition and gene structure organization, and evolutionary analysis for the duplicated pairs of POD. GO, KEGG, cis-regulatory elements, expression profiling of spatio-temporal response, and qRT-PCR analysis in response to (NaCl, drought, and ABA) disclosed extensive information on the gene functions and expression dynamics of tissue-specific and abiotic stress response in grapevine.\u003c/p\u003e\n\u003cp\u003eWe determined the phylogenetic relationships between grapevine (\u003cem\u003eVvPOD\u003c/em\u003e) and (\u003cem\u003eAtPOD\u003c/em\u003e) by comparative analysis. Results revealed an identical domain composition of \u003cem\u003eVvPOD\u003c/em\u003e with the model plant. The phylogenetic tree was categorized into 7 subgroups and the result of our tree are consistent with previously reported study of \u003cem\u003ePODs\u003c/em\u003e in Cassava [\u003ca href=\"#_ENREF_31\"\u003e31\u003c/a\u003e]. The motif composition analysis also demonstrated that motifs 1-4 are common among all the POD members with highly conserved nature. Moreover, the comparative structure analysis of POD showed that same subgroup shared a common junction. These results indicated a possible structural diversification within \u003cem\u003eVvPOD\u003c/em\u003e gene family, which plays an important role during the evolution of multi-gene family [\u003ca href=\"#_ENREF_32\"\u003e32\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eGene duplications are the vital force in the process of genomic evolution and functional divergence [\u003ca href=\"#_ENREF_33\"\u003e33\u003c/a\u003e]. Importantly, the gene duplication is considered a major component in the establishment of new genetic functions and evolutionary novelty [\u003ca href=\"#_ENREF_34\"\u003e34\u003c/a\u003e, \u003ca href=\"#_ENREF_35\"\u003e35\u003c/a\u003e]. Similarly, in the process of evolutionary history, most of the higher plant underwent polyploidization that is vital ingredient in shaping plant genome [\u003ca href=\"#_ENREF_36\"\u003e36\u003c/a\u003e]. In this study, the types of duplications in grapevine were identified by the help of MCScanX among \u003cem\u003ePOD\u003c/em\u003e genes. Results showed 4 types of duplications including, dispersed (20), tandem (15), segmental or whole-genome duplication (9), and proximal (3). It is noteworthy that during the process of evolution segmental and tandem duplication plays a critical role in the expansion of gene family [\u003ca href=\"#_ENREF_27\"\u003e27\u003c/a\u003e, \u003ca href=\"#_ENREF_37\"\u003e37\u003c/a\u003e, \u003ca href=\"#_ENREF_38\"\u003e38\u003c/a\u003e]. Though, in this study, we determined that both dispersed and tandem duplications contributed mainly to the expansion of \u003cem\u003ePOD\u003c/em\u003e gene family in grapevine. The selection pressure analysis are mainly based on three selection such as purifying (\u003cem\u003eKa/Ks\u003c/em\u003e\u0026lt;1), positive (\u003cem\u003eKa/Ks\u003c/em\u003e\u0026gt;1), and neutral selection (\u003cem\u003eKa/Ks\u003c/em\u003e=1), and its evolutionary assessment provide useful guidelines during the rate of divergence [\u003ca href=\"#_ENREF_39\"\u003e39\u003c/a\u003e]. Results inferred that 15 pairs of POD have shown less than 1.00 \u003cem\u003eKa/Ks\u003c/em\u003e ratio, indicating purifying selection and 7 pairs exhibiting more than 1.00 suggested the positive selection. The results of our study are also in consistent with previous reported studies on PODs [\u003ca href=\"#_ENREF_37\"\u003e37\u003c/a\u003e, \u003ca href=\"#_ENREF_40\"\u003e40\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eIn this study, we utilized the publically available RNA-seq data of 19 different tissues and organs to validate the tissue-specific response of 47 \u003cem\u003eVvPODs\u003c/em\u003e. Among them, 9 genes (\u003cem\u003eVvPOD1, VvPOD2, VvPOD6, VvPOD10, VvPOD12, VvPOD27, VvPOD32, VvPOD37, \u003c/em\u003eand\u003cem\u003e VvPOD46\u003c/em\u003e) exhibited significantly higher expression level among all the tissue and organs. The rest of the other genes showed down-regulation with similar tendency throughout grapevine tissues and organs developmental phases.\u003c/p\u003e\n\u003cp\u003eTo better understand the gene functions, expression profiling mainly provides a valuable clue to its regulatory role against specific treatment. Abiotic stresses considerably harbor plant growth and productivity, thus lead to economic losses. The drought severity hampered grapevine productivity and has deleterious impacts on viticulture worldwide [\u003ca href=\"#_ENREF_41\"\u003e41\u003c/a\u003e, \u003ca href=\"#_ENREF_42\"\u003e42\u003c/a\u003e]. Salt stress is one of the severe abiotic stress, while grapevine plants generally modify their physiology to combat salt stress severity [\u003ca href=\"#_ENREF_43\"\u003e43\u003c/a\u003e]. In regulating plant tolerance to several abiotic stress, an eminent hormone, such as ABA plays an essential role. Many researchers have indicated that plant peroxidases are involved in various cellular processes during plant growth and development, and its response to abiotic and biotic stress have been reported over the years [\u003ca href=\"#_ENREF_26\"\u003e26\u003c/a\u003e, \u003ca href=\"#_ENREF_44\"\u003e44\u003c/a\u003e]. For instance, membrane-bounded peroxidase (\u003cem\u003epmPOX1\u003c/em\u003e, \u003cem\u003epmPOX2a\u003c/em\u003e, \u003cem\u003epmPOX2b,\u003c/em\u003e and \u003cem\u003epmPOX3\u003c/em\u003e) in \u003cem\u003eZea\u003c/em\u003e \u003cem\u003emays \u003c/em\u003eroots are regulated by methyl jasmonate, salicylic acid and pathogen elicitors [\u003ca href=\"#_ENREF_21\"\u003e21\u003c/a\u003e]. Similarly, the overexpression of \u003cem\u003eCpPrx01\u003c/em\u003e altered the growth pattern of plants and condensed the level of a well-known hormone, indole-3-acetic acid (IAA) [\u003ca href=\"#_ENREF_45\"\u003e45\u003c/a\u003e]. Thus, in this study, the qRT-PCR validation of 30 \u003cem\u003ePOD\u003c/em\u003e genes in response to NaCl, drought, and ABA displayed a range of differential expression. The salt stress increased the expression of most \u003cem\u003ePOD\u003c/em\u003es, while drought and ABA stress decreased their expression. This suggested their important roles in grapevine against abiotic stresses.\u003c/p\u003e\n\u003cp\u003eTo improve and comprehend the possible transcriptional regulation functions of PODs, we also analyzed the GO, KEGG enrichment, and cis-elements in grapevine. Thus, these results inferred their diversified functions and indirect involvement in several biological processes, such as biotic-abiotic/hormone signaling.\u003c/p\u003e"},{"header":"4. Conclusions ","content":"\u003cp\u003eIn conclusion, we systematically identified a total of 47 \u003cem\u003ePOD\u003c/em\u003e genes in grapevine and were categorized into 7 subgroups, as supported by phylogenetic analysis. The GO, KEGG, and cis-elements analysis also extended our repositories on the diverse functions of PODs in plant developments during various stress-related activities in grapevine. While, the transcriptional profiling of various organs during several developmental stages and RT-PCR analysis, provide a stand-point of the PODs in improving the plant growth. Thus, the results of our study increase our understanding of \u003cem\u003ePOD\u003c/em\u003e genes in grapevine and laying the solid foundation for genetic improvement in other fruit crops.\u003c/p\u003e"},{"header":"5. Methods","content":"\u003cp\u003e\u003cem\u003e5.1\u003c/em\u003e \u003cem\u003eIdentification of POD Genes Family in Grapevine \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn order to identify the \u003cem\u003ePOD\u003c/em\u003e genes, we used the 73 reference sequences of \u003cem\u003eArabidopsis \u003c/em\u003eagainst grapevine (genome version 2.1) with the help of BioEdit tools. In general, we retrieved the sequences of grapevine and \u003cem\u003eArabidopsis\u003c/em\u003e from online sources such as ensembl (https://plants.ensembl.org/index.html) and TAIR (\u003ca href=\"http://www.arabidopsis.org/\"\u003ehttp://www.arabidopsis.org/\u003c/a\u003e). The domain composition was verified by using the NCBI-Conserved Domain database (\u003ca href=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/a\u003e) and SMART databases (\u003ca href=\"http://smart.embl-heidelberg.de/\"\u003ehttp://smart.embl-heidelberg.de/\u003c/a\u003e) [\u003ca href=\"#_ENREF_46\"\u003e46\u003c/a\u003e]. Those sequences with absent of POD domains and sequence with obvious error in length (\u0026gt;100aa length) were eliminated from the study before analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5.2 Phylogenetic Analysis of POD Gene Family\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor phylogenetic characterization, the multiple sequences alignment (MSA) of PODs was performed by MUSCLE [\u003ca href=\"#_ENREF_47\"\u003e47\u003c/a\u003e] using MEGA 7.0 software with the default options [\u003ca href=\"#_ENREF_48\"\u003e48\u003c/a\u003e]. The phylogenetic trees were constructed using the maximum likelihood (ML) method. After scrutiny of several models in MEGA 7.0, we choose the Jones, Taylor, and Thornton amino acid substitution model (JTT model) and for the reliability of resulting phylogenetic tree, the bootstrap values of 1000 replications were performed and keeping the other parameters as a default.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5.3 Calculation of Synonymous (Ks) and Non-synonymous (Ka) for Duplicated Genes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe rate of \u003cem\u003eKa/Ks\u003c/em\u003e was carried out for various types of duplicated pairs (i.e., dispersed, proximal, tandem, and segmental) by using MEGA 7.0 [\u003ca href=\"#_ENREF_48\"\u003e48\u003c/a\u003e]. The \u003cem\u003eKs\u003c/em\u003e and \u003cem\u003eKa\u003c/em\u003e ratio was intended by Nei- Gojobori method (Juke-Cantor) model in MEGA 7.0 with the bootstrap values of 1000 replicates. The various types of duplications of the \u003cem\u003ePOD\u003c/em\u003e gene pairs were detected by the MCScan algorithm.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5.4\u003c/em\u003e \u003cem\u003eGene Structure, Motifs Composition, and Physicochemical Analysis of POD\u003c/em\u003e \u003cem\u003eProtein \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor the gene structure illustration, we utilized the GFF3 file of the grapevine genome and images were implemented by TBtools software [\u003ca href=\"#_ENREF_49\"\u003e49\u003c/a\u003e]. The motifs analysis of POD protein were performed by the Multiple Em of Motif Elicitation (MEME Suite) version 5.0.5 and then demonstrated by TBtools software. The following parameter was calibrated for this purpose as follows: the maximum number of motifs 10, with a minimum width of 50 and a maximum of 100 and the other parameter was set as default [\u003ca href=\"#_ENREF_50\"\u003e50\u003c/a\u003e]. While, for each gene of POD, the several physicochemical properties (i.e., molecular weight (MW), isoelectronic points (PIs), and GRAVY) were intended by ExPASY PROTPARAM tools (\u003ca href=\"http://web.expasy.org/protparam/\"\u003ehttp://web.expasy.org/protparam/\u003c/a\u003e). The subcellular localization was further predicted by using the WOLF PSORT online server (\u003ca href=\"https://wolfpsort.hgc.jp/\"\u003ehttps://wolfpsort.hgc.jp/\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5.\u003c/em\u003e5 \u003cem\u003eGene Ontology (GO), Kyoto Encyclopedia of Genes and Genomics (KEGG) and Cis-Elements Predictions of Class III Peroxidase Gene Family\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor the GO and KEGG enrichment analysis, the online panther server (\u003ca href=\"http://pantherdb.org/\"\u003ehttp://pantherdb.org/\u003c/a\u003e) and the genome server was implemented (https://www.genome.jp/kegg/pathway.html) and their enriched pathways were further explored by TBtools software [\u003ca href=\"#_ENREF_49\"\u003e49\u003c/a\u003e]. The POD promoter sequences (i.e., selected as 1500bp) was initially imported in Generic File Format (GFF) from the grapevine genome. After that various cis-regulatory elements for each promoter sequence was identified by the PlantCARE database (\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/a\u003e) [\u003ca href=\"#_ENREF_51\"\u003e51\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5.6\u003c/em\u003e \u003cem\u003eChromosomal Mapping and Gene Collinearity Analysis of Grapevine and Arabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe grapevine genomic database (CRIBI. Available online: http://genomes.cribi.unipd.it/grape/, V2.1), was utilized for the chromosomal locations of \u003cem\u003ePOD\u003c/em\u003e genes and were mapped based on information available. Similarly, for gene collinearity analysis, the grapevine and \u003cem\u003eArabidopsis \u003c/em\u003erelationship was verified and demonstrated with the help of Circos (TBtools software) program was applied [\u003ca href=\"#_ENREF_49\"\u003e49\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5.7\u003c/em\u003e \u003cem\u003ePlant Material and Methods\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, we used the two-year-old potted grapevine plants (\u003cem\u003eV. vinifera\u003c/em\u003e cv. Summer Black), selected from greenhouse condition (25\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026thinsp;\u0026deg;C) under 16-h light/8-h dark photoperiod and 65% relative humidity (RH) at the Nanjing Agricultural University Nanjing-China. The grapevine plants were subjected to abiotic stress including, NaCl (100 mM), drought (irrigated with 15% (w/v) polyethylene glycol), and ABA (100 \u0026mu;M/100 mL of water) with an interval of 1, 6, 24 h against control (CK), with three biological replicates and the three samples were mixed to make one composite sample while using the 4th unfolded leaf from cv. Summer Black grapevine. At last, all the samples were quickly frozen in liquid nitrogen and stored at \u0026minus;80 \u0026deg;C for further use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5.8\u003c/em\u003e \u003cem\u003eRNA Isolation and Transcriptional Profiling of POD Gene Family in Grapevine\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the grapevine leaves with the help of Trizol (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. Then, the Primer Script RT reagent kit (TAKARA, Dalian, China) was used to convert the RNA into reverse-transcribed into cDNA. The gene-specific primers (Supplementary Table S6) was designed by the help of Becan Designer 7.9 and their specificity was confirmed using the BLAST tool against the grapevine genome. Also, we performed the RT-PCR by following the previously reported studies [\u003ca href=\"#_ENREF_52\"\u003e52\u003c/a\u003e, \u003ca href=\"#_ENREF_53\"\u003e53\u003c/a\u003e], and the relative fold expression was calculated using the comparative Ct-method. The expression patterns of all \u003cem\u003ePOD\u003c/em\u003e gene were analyzed based on a previous study [\u003ca href=\"#_ENREF_38\"\u003e38\u003c/a\u003e, \u003ca href=\"#_ENREF_54\"\u003e54\u003c/a\u003e], and for reference gene in qRT- PCR, we used the housekeeping actin gene (\u003cem\u003eAB073011\u003c/em\u003e). In summary, the real-time PCR amplification reactions were performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, CA, USA) using SYBR Green (Applied Biosystems, CA, USA) with three replicates. While the following PCR parameter conditions were set as follow: denaturation at 95\u0026thinsp;\u0026deg;C for 2\u0026thinsp;min, 40 cycles of denaturation at 95\u0026thinsp;\u0026deg;C for 10\u0026thinsp;s, annealing at 60\u0026thinsp;\u0026deg;C for 40\u0026thinsp;s, and extension at 72\u0026thinsp;\u0026deg;C for 15\u0026thinsp;s.\u003c/p\u003e\n\u003cp\u003eFor expression profiling, we utilized the RNA-sequence data from the NCBI GEO website (https://www.ncbi.nlm.nih.gov/geo/) under the series entry GSE36128. Further, the expression levels were quantified by FPKM (fragments per kilobase of transcript per million fragments mapped), and heat maps were generated by using Rstudio (R program package) based on log\u003csub\u003e2\u003c/sub\u003e FPKM values.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePODs: peroxidase\u003c/p\u003e\n\u003cp\u003eABA: Abscisic acid\u003c/p\u003e\n\u003cp\u003eCSD: coding sequence\u003c/p\u003e\n\u003cp\u003eMW: molecular weight\u003c/p\u003e\n\u003cp\u003ePIs: isoelectric point\u003c/p\u003e\n\u003cp\u003eGRAVY: grand average of hydropathicity\u003c/p\u003e\n\u003cp\u003eWGD: the whole genome duplication\u003c/p\u003e\n\u003cp\u003eGO: Gene Ontology Enrichment\u003c/p\u003e\n\u003cp\u003eKEGG: Kyoto Encyclopedia of Genes Genomics\u003c/p\u003e\n\u003cp\u003eMF: Molecular Functions\u003c/p\u003e\n\u003cp\u003eCC: Cellular Component\u003c/p\u003e\n\u003cp\u003eBP: biological process\u003c/p\u003e\n\u003cp\u003ePCC: Pearson\u0026rsquo;s Correlation Coefficient\u003c/p\u003e\n\u003cp\u003eFPKM: Fragments per kilobase of transcript per million fragments mapped\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: Not applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: All data generated or analysed during this study are included in this published article and its supplementary information files\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding: This study was financially supported by The National Key Research and Development Project (2018YFD1000200), The Key Research and Development Project of Jiangsu Province (BE2018389), Jiangsu Planned Projects for Postdoctoral Research Funds, National Natural Science Foundation of China (31801809, 31872047).\u003c/p\u003e\n\u003cp\u003eAuthors' contributions: H. X., C. W., L. G., X. L. conceived and designed the experiments, H. X., C. W. performed the experiment: N. K., M. C., W. F. analyzed the data: L.G., X. L., wrote the manuscript. All authors have read and approved the manuscript\u003c/p\u003e\n\u003cp\u003eAcknowledgements: Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWelinder KG: \u003cstrong\u003eSuperfamily of plant, fungal and bacterial peroxidases\u003c/strong\u003e. \u003cem\u003eCurrent Opinion in Structural Biology \u003c/em\u003e1992, \u003cstrong\u003e2\u003c/strong\u003e(3):388-393.\u003c/li\u003e\n\u003cli\u003eMath\u0026eacute; C, Barre A, Jourda C, Dunand C: \u003cstrong\u003eEvolution and expression of class III peroxidases\u003c/strong\u003e. \u003cem\u003eArchives of Biochemistry and Biophysics \u003c/em\u003e2010, \u003cstrong\u003e500\u003c/strong\u003e(1):58-65.\u003c/li\u003e\n\u003cli\u003eHiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H: \u003cstrong\u003eA large family of Class III plant peroxidases\u003c/strong\u003e. \u003cem\u003ePlant Cell Physiol \u003c/em\u003e2001, \u003cstrong\u003e42\u003c/strong\u003e(5):462-468.\u003c/li\u003e\n\u003cli\u003eAlmagro L, G\u0026oacute;mez Ros LV, Belchi-Navarro S, Bru R, Ros Barcel\u0026oacute; A, Pedre\u0026ntilde;o MA: \u003cstrong\u003eClass III peroxidases in plant defence reactions\u003c/strong\u003e. \u003cem\u003eJournal of Experimental Botany \u003c/em\u003e2008, \u003cstrong\u003e60\u003c/strong\u003e(2):377-390.\u003c/li\u003e\n\u003cli\u003eFrancoz E, Ranocha P, Nguyen-Kim H, Jamet E, Burlat V, Dunand C: \u003cstrong\u003eRoles of cell wall peroxidases in plant development\u003c/strong\u003e. \u003cem\u003ePhytochemistry \u003c/em\u003e2015, \u003cstrong\u003e112\u003c/strong\u003e:15-21.\u003c/li\u003e\n\u003cli\u003eGabald\u0026oacute;n C, L\u0026oacute;pez-Serrano M, Pedre\u0026ntilde;o MA, Barcel\u0026oacute; AR: \u003cstrong\u003eCloning and molecular characterization of the basic peroxidase isoenzyme from \u003cem\u003eZinnia elegans\u003c/em\u003e, an enzyme involved in lignin biosynthesis\u003c/strong\u003e. \u003cem\u003ePlant Physiology \u003c/em\u003e2005, \u003cstrong\u003e139\u003c/strong\u003e(3):1138-1154.\u003c/li\u003e\n\u003cli\u003eDaudi A, Cheng Z, O\u0026rsquo;Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP: \u003cstrong\u003eThe apoplastic oxidative burst peroxidase in \u003cem\u003eArabidopsis\u003c/em\u003e is a major component of pattern-triggered immunity\u003c/strong\u003e. \u003cem\u003eThe Plant cell \u003c/em\u003e2012, \u003cstrong\u003e24\u003c/strong\u003e(1):275-287.\u003c/li\u003e\n\u003cli\u003eBindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C, Davies DR\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003ePeroxidase-dependent apoplastic oxidative burst in \u003cem\u003eArabidopsis\u003c/em\u003e required for pathogen resistance\u003c/strong\u003e. \u003cem\u003eThe Plant Journal \u003c/em\u003e2006, \u003cstrong\u003e47\u003c/strong\u003e(6):851-863.\u003c/li\u003e\n\u003cli\u003eLlorente F, Lopez-Cobollo RM, Catala R, Martinez-Zapater JM, Salinas J: \u003cstrong\u003eA novel cold-inducible gene from \u003cem\u003eArabidopsis\u003c/em\u003e, RCI3, encodes a peroxidase that constitutes a component for stress tolerance\u003c/strong\u003e. \u003cem\u003eThe Plant journal : for cell and molecular biology \u003c/em\u003e2002, \u003cstrong\u003e32\u003c/strong\u003e(1):13-24.\u003c/li\u003e\n\u003cli\u003eShigeto J, Tsutsumi Y: \u003cstrong\u003eDiverse functions and reactions of class III peroxidases\u003c/strong\u003e. \u003cem\u003eNew Phytologist \u003c/em\u003e2016, \u003cstrong\u003e209\u003c/strong\u003e(4):1395-1402.\u003c/li\u003e\n\u003cli\u003ePassardi F, Tognolli M, De Meyer M, Penel C, Dunand C: \u003cstrong\u003eTwo cell wall associated peroxidases from \u003cem\u003eArabidopsis\u003c/em\u003e influence root elongation\u003c/strong\u003e. \u003cem\u003ePlanta \u003c/em\u003e2006, \u003cstrong\u003e223\u003c/strong\u003e(5):965-974.\u003c/li\u003e\n\u003cli\u003eCosio C, Dunand C: \u003cstrong\u003eSpecific functions of individual class III peroxidase genes\u003c/strong\u003e. \u003cem\u003eJournal of Experimental Botany \u003c/em\u003e2008, \u003cstrong\u003e60\u003c/strong\u003e(2):391-408.\u003c/li\u003e\n\u003cli\u003eHerrero J, Fernandez-Perez F, Yebra T, Novo-Uzal E, Pomar F, Pedreno MA, Cuello J, Guera A, Esteban-Carrasco A, Zapata JM: \u003cstrong\u003eBioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis\u003c/strong\u003e. \u003cem\u003ePlanta \u003c/em\u003e2013, \u003cstrong\u003e237\u003c/strong\u003e(6):1599-1612.\u003c/li\u003e\n\u003cli\u003eHeggie L, Jansen MA, Burbridge EM, Kavanagh TA, Thorneley RN, Dix PJ: \u003cstrong\u003eTransgenic tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e L. cv. 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Roman\",serif;color:black;margin-top:6.0pt;text-indent:21.85pt;'\u003e\u003cspan style=\"font-family: Helvetica; color: rgb(0, 0, 0); font-size: 12px;\"\u003eTable.1. The \u003cem\u003ePOD\u003c/em\u003e genes in grapevine with outlier \u003cem\u003eKa/Ks\u003c/em\u003e and various types of duplications of the \u003cem\u003ePOD\u003c/em\u003e gene pairs with the detection by\u003c/span\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e the MCScan algorithm (i.e., Dispersed, proximal, tandem, and segmental).\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eGene 1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eGene 2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eKs\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eKa\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eKa\u003c/em\u003e/\u003cem\u003eKs\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eSelection Pressure\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eGene Duplications\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD1\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD2\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.76\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.65\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.86\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD3\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD5\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.69\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.58\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.83\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD9\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD10\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.68\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.63\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.92\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD11\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD14\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.89\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.65\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.73\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New 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style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD16\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.04\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.01\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.28\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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0);\"\u003e\u003cem\u003eVvPOD18\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.41\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.11\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.08\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD25\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD26\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.92\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.62\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.68\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD27\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD30\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.56\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.74\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.33\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePositive Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD31\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD32\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.60\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.64\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.07\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePositive Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD42\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD43\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.95\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.36\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.38\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eDispersed\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD24\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD35\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.75\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.43\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.58\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eProximal\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD4\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD12\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.20\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.45\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.38\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eTandem\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD13\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD19\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.50\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.53\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.05\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePositive Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eTandem\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD20\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD21\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.18\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.04\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.24\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eTandem\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD22\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD23\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.24\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.56\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e2.35\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePositive Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eTandem\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD33\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD34\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.40\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.05\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.13\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eTandem\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD38\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD36\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.20\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.09\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.47\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eTandem\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD40\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD41\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.07\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.03\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.35\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePurifying Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eTandem\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD6\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD7\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.22\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.37\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.68\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePositive Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eWGD or Segmental\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD8\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD28\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.57\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.67\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.19\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePositive Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eWGD or Segmental\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD29\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54.05pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cem\u003eVvPOD36\u003c/em\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 40.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.48\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e0.61\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 42.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e1.28\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106.3pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003ePositive Selection\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101.75pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eWGD or Segmental\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style='margin:0in;margin-bottom:.0001pt;text-align:justify;line-height:17.0pt;font-size:16px;font-family:\"Times New Roman\",serif;color:black;margin-top:6.0pt;'\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003eWGD or Segmental\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cspan style=\"font-family: \n Helvetica;\"\u003e\u003cspan style=\"font-size: \n 12px;\"\u003e\u003cspan style=\"color: rgb(0, 0, 0);\"\u003e\u003cbr\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;margin-bottom:.0001pt;text-align:left;line-height:normal;font-size:16px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u003cspan style=\"font-size: 12px; font-family: Helvetica; color: rgb(0, 0, 0);\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"},{"header":"Supplementary Legends","content":"\u003cp\u003eSupplementary Figure:\u003c/p\u003e\n\u003cp\u003eFigure S1.The LOGOS of PODS were also elucidated by MEME online server.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupplementary Tables:\u003c/p\u003e\n\u003cp\u003eTable S1. The basic information of PODsgenes identified in grapevine.\u003c/p\u003e\n\u003cp\u003eTable S2. The gene ontology (GO) of POD genes in grapevine.\u003c/p\u003e\n\u003cp\u003eTable S3. Kyoto Encyclopedia of Genes and Genomics (KEGG) of POD genes in grapevine.\u003c/p\u003e\n\u003cp\u003eTable S4. Cis-Elements of PODgenes in grapevine.\u003c/p\u003e\n\u003cp\u003eTable S5. Relative expression patterns (log2) of POD genes in grapevine by qRT-PCR.\u003c/p\u003e\n\u003cp\u003eTable S5S6.Sequences of the PODgenes primers used for quantitative real-time PCR.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Grapevine; Genome-wide analysis; POD genes family; Collinearity and Expression analysis","lastPublishedDoi":"10.21203/rs.2.16423/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.16423/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Background : The class III peroxidases (PODs) are involved in a broad range of physiological activities, such as the formation of lignin, cell wall components, defense against pathogenicity or herbivore, and abiotic stress tolerance. The POD family members have been well-studied and characterized by bioinformatics analysis in several plant species, but no previous genome-wide analysis has been carried out of this gene family in grapevine to date.\nResults : We comprehensively identified 47 PODs in the grapevine genome and are further classified into 7 subgroups based on their phylogenetic analysis. Results of motif composition and gene structure organization analysis revealed that PODs in the same subgroup shared similar conjunction while the protein sequences were highly conserved. Intriguingly, the integrated analysis of chromosomal mapping and gene collinearity analysis proposed that both dispersed and tandem duplication events contributed to the expansion of PODs in grapevine. Also, the gene duplication analysis suggested that most of the genes (20) were dispersed followed by (15) tandem, (9) segmental or whole-genome duplication, and (3) proximal, respectively. The evolutionary analysis of PODs, such as Ka/Ks ratio of the 15 duplicated gene pairs were less than 1.00, indicated that most of the gene pairs exhibiting purifying selection and 7 pairs underwent positive selection with value greater than 1.00. The Gene Ontology Enrichment (GO), Kyoto Encyclopedia of Genes Genomics (KEGG) analysis, and cis-elements prediction also revealed the positive functions of PODs in plant growth and developmental activities, and response to stress stimuli. Further, based on the publically available RNA-sequence data, the expression patterns of PODs in tissue-specific response during several developmental stages revealed diverged expression patterns. Subsequently, 30 genes were selected for RT-PCR validation in response to (NaCl, drought, and ABA), which showed their critical role in grapevine.\nConclusions : In conclusion, we predict that these results will lead to novel insights regarding genetic improvement of grapevine.","manuscriptTitle":"Genome-wide Identification of the Class III POD Gene Family and their Expression Profiling in Grapevine (Vitisvinifera L)","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2020-04-02 14:59:34","doi":"10.21203/rs.2.16423/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-04-24T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-04-23T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-04-01T12:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-04-01T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewersInvited","content":"","date":"2020-03-31T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-03-31T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-03-30T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-03-29T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-03-29T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2019-10-24 22:39:32","doi":"10.21203/rs.2.16423/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-02-27T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-02-15T12:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2019-12-16T12:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2019-11-13T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewersInvited","content":"","date":"2019-10-24T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2019-10-24T12:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2019-10-18T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-10-18T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2019-10-17T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2019-10-15T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d7e19b73-cf85-480f-b55a-77a7d2caedd3","owner":[],"postedDate":"April 2nd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33462,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2020-07-05T15:01:09+00:00","versionOfRecord":{"articleIdentity":"rs-7066","link":"https://doi.org/10.1186/s12864-020-06828-z","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2020-06-29 12:00:00","publishedOnDateReadable":"June 29th, 2020"},"versionCreatedAt":"2020-04-02 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