Genetic variability of Grapevine Pinot gris virus and its association with grapevine leaf mottling and deformation

Phytopathology, Saldarelli 1

Genetic variability of Grapevine Pinot gris virus and its association with grapevine leaf 1

mottling and deformation 2

3

P. Saldarelli, A. Giampetruzzi, M. Morelli, U. Malossini1, C. Pirolo, P. Bianchedi

1, V. Gualandri

1 4

CNR Istituto per la Protezione Sostenibile delle Piante, UOS-Bari, and Dipartimento di Scienze del 5

Suolo della Pianta e degli Alimenti, Università degli Studi di Bari via Amendola 165/A, 70126, 6

Bari, Italy; 7

1FEM-IASMA, Centre for Technology Transfer, via E. Mach 1, 38010, San Michele all’Adige 8

(Trento), Italy. 9

10

Corresponding author 11

Dr. Pasquale Saldarelli 12

Istituto per la Protezione Sostenibile delle Piante (CNR-IPSP), UOS Bari 13

Via Amendola 122/D, 70126 Bari, Italy 14

Tel. 0039.0805443065 Fax. 0039.0805443608 15

E-mail: [email protected] 16

17

18

ABSTRACT 19

The role of Grapevine Pinot gris virus (GPGV) in the etiology of grapevine leaf mottling and 20

deformation was investigated by biological and molecular assays. A survey on different cultivars 21

from the Trentino Region in Italy showed a widespread distribution of GPGV, which was 22

associated with symptomatic (79%) but also with symptomless vines (21%). Symptomatic and 23

GPGV-infected Pinot gris vines induced symptoms on grafted vines of healthy Pinot gris or 24

Traminer, whereas GPGV-infected but symptomless vines did not. High-throughput sequencing of 25

small RNA (sRNAs) populations of two infected Pinot gris accessions confirmed the existence of 26

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nearly overlapping viromes in vines with or without symptoms but phylogenetic analyses of the 27

genomes of seven GPGV isolates from Italy and the Czech and Slovak Republics clearly 28

differentiated those infecting symptomatic vines. The involvement of Grapevine rupestris vein 29

feathering virus (GRVFV) in the disease, which was only infecting the symptomatic vine, was ruled 30

out by RT-PCR studies. Maximum likelihood and Bayesian phylogenetic analysis of two GPGV 31

genomic regions, encompassing part of the movement protein and coat protein gene sequences 32

(MP/CP) and the RNA dependent RNA polymerase domain (RdRp) of the replicase gene, showed 33

that isolates from symptomatic vines form a lineage distinct from that of symptomless vines. 34

Moreover, the presence or lack of the MP stop codon identified in viral isolates from symptomatic 35

or symptomless vines, respectively, is likely responsible for a six amino acids longer MP in 36

symptomless isolates. 37

38

P. Saldarelli, A. Giampetruzzi, M. Morelli, U. Malossini, C. Pirolo, P. Bianchedi, V. Gualandri, 39

2014, Genetic variability of Grapevine Pinot gris virus and its association with grapevine leaf 40

mottling and deformation, Phytopathology 41

42

43

Symptoms of stunting, chlorotic mottling and leaf deformation had been observed on Vitis 44

vinifera cv. Pinot gris, in Trentino vineyards since 2003. After several clostero-, ampelo-, nepo- and 45

vitiviruses were excluded as possible causes for these symptoms, a metagenomic investigation, 46

using a NGS (Next Generation Sequencing) approach, was undertaken to determine the etiology. 47

This led to the identification of a new trichovirus, Grapevine Pinot gris virus (GPGV), whose full-48

length sequence was described and shown to be closely related to that of Grapevine berry inner 49

necrosis virus (GINV) (7). Similar symptoms were later reported from Emilia Romagna 50

(http://archives.eppo.int/EPPOReporting/2014/Rsf-1401.pdf) in cv. Chardonnay, from Trentino (14) 51

and Friuli Venezia Giulia (13) from cvs. Traminer and Pinot noir and from Apulia in the table grape 52

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cvs. Black Magic and Supernova (18). The virus was also detected in Veneto (22) in cv. Glera, and 53

in Slovenia (20), in cvs. Pinot gris, Sauvignonasse and Muscat blanc. In Slovenia GPGV-like 54

symptoms are widely spread, 40 out of 42 symptomatic vines (all which tested negative from the 55

main grapevine nepoviruses) being infected by GPGV (20). Outside of Europe, GPGV was found in 56

Korea (2) on the table grape cv. Tamnara, which showed symptoms of berry necrosis similar to 57

those described for the related virus GINV (27). GPGV was also detected in the Czech and Slovak 58

Republics, where the complete genome of three additional isolates was determined (8). These 59

isolates have a low nucleotide sequence heterogeneity and differ from the Italian isolate because of 60

localized divergences within the ORF1 amino acid sequence, which could result from events of 61

recombination with GINV. In addition, a random survey on a set of Slovak and Czech grapevine 62

accessions resulted in GPGV detection in 13 out of 58 vines but the specific symptoms associated 63

with the presence of the virus were not observed since these vines hosted multiple viruses (8). 64

A three year field study showed that vines affected by the GPGV-associated disease had fewer 65

canes and a lower number and weight of the bunches (14). 66

All mentioned reports show that, wherever it occurs in Europe, the association of GPGV with 67

symptoms is contradictory, a feature noticed since its discovery (7), that disclosed the frequent 68

presence of a latent behavior. 69

In the present study a survey was carried out on a group of 92 GPGV isolates from Trentino to 70

assess the virus/symptom association. Biological indexing of symptomless and symptomatic Pinot 71

gris vines was performed and the viromes of two vines, with or without symptoms, were analyzed 72

by sequencing their sRNAs. This allowed the assembling of GPGV genome consensus sequences, 73

which were phylogenetically compared with full-length genomes available in databases. Moreover, 74

the genetic diversity among GPGV isolates was investigated in two genome regions, the first 75

encompassing part of the movement protein and the coat protein (MP/CP) sequences, the second 76

corresponding to the RNA dependent RNA polymerase domain (RdRp) of the replicase gene. 77

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Collectively, the results suggest that GPGV exists as a population of genetically distinct virulent 78

and latent isolates. 79

80

MATERIALS AND METHODS 81

82

Plant sources and grafting assays In this study 100 grapevine accessions were analyzed 83

(Table 1), 92 of which, originating from different Trentino vineyards, were selected based on the 84

presence or absence of symptoms. Pinot gris accessions ZA505-1A and ZA505-2N were previously 85

investigated by Giampetruzzi et al. (7), whereas ZA505-2A (symptomatic) and ZA505-1N 86

(symptomless) were the source of the sRNAs populations analysed in the present work. Dormant 87

canes were collected from these vines from 2010 to 2014 and from infected table grape cultivars 88

Black Magic and Supernova (18). 89

Biological assays were performed by bud- and green-grafting (26). During 2009, dormant 90

cuttings from symptomatic or symptomless Pinot gris vines were rooted in pots, then grafted with 91

buds from healthy Vitis rupestris “St. George” and Vitis vinifera cv. Cabernet franc, Pinot gris and 92

Traminer. After one year, successful grafts were transferred to the field and symptoms scored in 93

2010 and 2014. Green-grafting was made in 2014 as described by Taylor et al. (26). Grafted plants 94

were maintained in a greenhouse and scored for symptoms in the same year. 95

RNA extraction, RT-PCR and synthesis of sRNA libraries. For GPGV detection by RT-96

PCR, total RNAs were purified from 100 mg of cortical scrapings using the RNeasy Plant Mini Kit 97

(Qiagen, the Netherlands) as described by MacKenzie et al. (12). Detection of relevant grapevine 98

viruses associated with leafroll, (Grapevine leafroll associated virus-1, GLRaV-1; Grapevine 99

leafroll associated virus-2, GLRaV-2; Grapevine leafroll associated virus-3, GLRaV-3), rugose 100

wood (Grapevine virus A, GVA and Grapevine virus B, GVB), infectious degeneration (Grapevine 101

fanleaf virus, GFLV; Arabis mosaic virus, ArMV) and Grapevine fleck virus (GFkV) was done by 102

multiplex RT-PCR as reported (6), whereas primers and the protocol for Grapevine rupestris stem 103

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pitting associated virus (GRSPaV) were described by Meng et al. (17). DNA fragments 104

corresponding to part of the MP/CP and RdRp gene domains were amplified from total RNA after 105

random-primed reverse transcription by Moloney murine leukemia virus reverse transcriptase 106

(ThermoFisher Scientific, USA). Resulting cDNA was subjected to PCR with two sets of GPGV 107

primers selected by Primer-BLAST, which allows the evaluation of unspecific matching to the Vitis 108

vinifera genome. Primers designed on the 3’end of the movement protein and the 5’ end of the viral 109

coat protein (MP/CP) gene sequences, DetF (5’-TGGTCTGCAGCCAGGGGACA-3’) and DetR 110

(5’-TCACGACCGGCAGGGAAGGA-3’), were already described (18), whereas GPGVRepF (5’-111

TGAGGCATTCGATGTTTCCCA-3’) and GPGVRepR (5’-ACCCAATCAAGCCATGAACCT-112

3’) were designed to target the RdRp domain of the GPGV replicase gene. For both sets of primers, 113

PCR was performed in 1X PCR buffer containing 2.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM each 114

primer, 0.0375 U Dream Taq DNA polymerase (ThermoFisher Scientific, USA) and the following 115

cycling conditions: initial denaturation at 94°C for 2 minutes , followed by 30 cycles at 94°C for 30 116

seconds, 60°C for 40 seconds and 72°C for 45 seconds, and a final extension at 72°C for 7 minutes. 117

Grapevine rupestris vein feathering virus (GRVFV) was detected by RT-PCR with four different 118

sets of primers: VF-F 5’-CGAAGCTCACTGGCGGACTTCTG-3’, VF-R 5’-119

GGCACAGAAGCCAAGGCGTTCA-3 (S. Sabanadzovic, personal communication); C105F1 5’-120

CCTGTCGCTTCCTTCTCATCT-3’, C105R2 5’-CATCTTCCATGCCCATTTCTTG-3’ (M. Al 121

Rwahnih, personal communication); GRVFV2801 5’-CCTTGACTGCCTCCTCGTCTC-3’, 122

GRVFV3506 5’-TCCGAGTCTCCAGCGATCAGC-3’ (our sequences, unpublished); GRVFV5202 123

5’-GATGACAACACCGATTACAAC-3’, GRVFV6439 5’-AATGCCCATTGGGCCAGAGAC-3’ 124

(our sequences, unpublished). Amplicons were visualized by standard 1.2 % agarose gel 125

electrophoresis. DNA fragments corresponding to the MP/CP and RdRp amplicons were ligated to 126

the pSC-A amp/kan vector and cloned in Escherichia coli SoloPack competent cells (StrataClone 127

PCR cloning kit, Agilent Technologies, USA). At least three independent cDNA clones per isolate 128

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were custom-sequenced (Macrogen Europe, The Netherlands). Preliminary DNA data analysis was 129

done with SerialCloner 2.6 (http://serialbasics.free.fr/Serial_Cloner.html). 130

Small RNAs, extracted from 1 g of leaf and petiole tissues of accessions ZA505-2A and 131

ZA505-1N following selective PEG-precipitation from total RNAs, were used for the synthesis of 132

libraries as described by Giampetruzzi et al. (7). Libraries were sequenced on an Illumina 133

HiScanSQ apparatus in 50 nt-long single-read runs. 134

Bioinformatic analysis. Small RNA libraries were pre-processed using the UEA sRNA toolkit 135

workbench software (23) to trim the 3' adapter, remove low quality reads and filter out reads 136

matching known plant tRNA or rRNA sequences (Rfam version 10, Jan-2010). Filtering of reads 137

aligning to the Vitis vinifera genome was done by the Patman program (21) allowing no nucleotide 138

mismatches. Contigs were generated by the de novo-assembly software Velvet 1.2.08 (28) using k-139

mer values of 15 and 17. Contig homologies with virus and viroid sequences were obtained by 140

BLASTX and BLASTN (1) search using a virus and viroid RefSeq database 141

(http://www.ncbi.nlm.nih.gov/refseq/) with e-values thresholds of 10-6 and 10

-4, respectively. Short 142

read alignment toward selected virus and viroid genome sequences [GPGV IT GenBank accession 143

no. NC_015782.1; GPGV SK01, GenBank accession no. 543887404; GPGV SK13 GenBank 144

accession no. 543887408; GPGV SK30, GenBank accession no. 543887400; GRVFV GenBank 145

accession no. 57116481; GRSPaV GenBank accession 9630737; Grapevine yellow speckle viroid-1 146

(GYSVd-1) GenBank accession no. 323482824; Hop stunt viroid (HSVd) GenBank accession 147

no.11497495] was made with SOAP software (11), allowing up to two nucleotide mismatches. 148

Whole genome consensus sequences from GPGV ZA505-2A, ZA505-1N and ZA505-2N (7) 149

isolates (ZA505-2Acons, ZA505-1Ncons and ZA505-2Ncons) were assembled with Geneious 7.0 150

(Biomatters Limited, New Zealand) on the .sam file generated by SOAP and on the GPGV IT or 151

GPGV SK30 reference sequences. Positions with zero coverage (i.e. not covered by any reads) were 152

indicated by the character N, whereas the most frequent base resulting from the alignment of reads 153

to the reference sequence was used (plurality rule). 154

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Consensus sequences from the MP/CP- and RdRp-cloned regions, were obtained by the 155

multiple alignment tools of Geneious 7.0 using at least three different recombinant plasmids for 156

each isolate. Nucleotide sequences were aligned with MUSCLE (4) as implemented in MEGA 157

5.2.2 (24). The evaluation of the best model of nucleotide substitution, phylogenetic tree analysis 158

with the maximum likelihood method and the estimation of the statistical significance of branch 159

order by 1000 bootstrap re-sampling of the original alignment, were inferred by the same software 160

package. Sequences were analysed for recombination events by the RDP3 package (16). The 161

existence of a temporal signal in the MP/CP sequence was assessed by Path-O-Gen 1.4 software 162

(http://tree.bio.ed.ac.uk/software/pathogen/). Reliability of phylogenetic reconstruction was 163

assessed by evaluating Bayesian posterior probabilities with BEAST 1.8.0 (3) using the Hasegawa-164

Kishino-Yano (HKY) substitution model (9) with a 4-category discrete approximation of a gamma 165

distribution (HKY+ Γ) to take into account among-site heterogeneity, and a relaxed uncorrelated 166

molecular clock. Convergence of all parameters was achieved by running 5x108 chains until 167

obtaining an Effective Sample Size (ESS) >200. Posterior probabilities and major evolutionary 168

parameters were estimated by TRACER (http://tree.bio.ed.ac.uk/software/tracer). Maximum clade 169

credibility tree (MCC) was obtained by TREEANNOTATOR (beast.bio.ed.ac.uk), discarding the 170

10% of trees, whereas FIGTREE (http://tree.bio.ed.ac.uk/software/figtree/) allowed graphical 171

display of the BEAST-generated trees. 172

173

RESULTS 174

175

Biological indexing. Indicators V. vinifera cv. Cabernet franc and Vitis rupestris did not show 176

symptoms of leaf mottling and deformation during four years of observations when grafted on 177

either symptomatic or symptomless Pinot gris vines (Table 2). Conversely, when cvs. Pinot gris or 178

Traminer were used as indicators, symptoms were observed the first year after grafting in almost all 179

grafted combinations in which a symptomatic vine was tested. No symptoms were observed when 180

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symptomless Pinot gris vines were indexed on Pinot gris and Traminer (Table 2). It should be 181

pointed out that these assays were initiated in 2009 when GPGV was not yet discovered; that all 182

tested vines, either symptomless or symptomatic in the field, were infected by GPGV (Table 1 and 183

2), and that the virus was always transmitted by grafting, as assessed by RT-PCR. By contrast, the 184

Pinot gris and Traminer vines used as indicators tested negative for GPGV by RT-PCR. Additional 185

green-grafting assays were performed in 2014 using GPGV-infected vines ZA505-1A, ZA505-2A, 186

ZA505-1N and ZA505-2N (Table 1), whose sanitary status was defined by NGS analysis of sRNAs 187

populations. Results confirmed bud-grafting assays, for symptoms developed only on indicators 188

grafted on symptomatic vines (Table 2). These findings suggested either that GPGV is not 189

involved in the induction of symptoms, thus an unknown pathogen is the etiological agent of the 190

disease, or that virulent and latent strains of GPGV exist. 191

The viromes of GPGV symptomatic and symptomless vines. To investigate the possible 192

existence of unknown viral or viroid pathogens associated with disease symptoms, sRNA libraries 193

from two additional Pinot gris vines, named ZA505-1N and ZA505-2A, were constructed and 194

analyzed. Symptomless (ZA505-1N) or symptomatic (ZA505-2A) leaves with petioles were 195

collected in the period of optimal symptom expression (spring 2013). After removing low 196

complexity and t/rRNA sequences, 1,970,760 and 1,933,512 unique reads 16-35 nucleotide-long 197

were obtained from vines ZA505-1N and ZA505-2A, respectively. These corresponded to 198

8,756,149 (ZA505-1N) and 7,823,623 (ZA505-2A) redundant reads (not shown) with a profile 199

distribution showing the predominance of 21 nt-small RNAs and 21/24 nt ratios of 1.75 and 2.32 in 200

ZA505-1N and ZA505-2A, respectively (Supplementary Figure 1). Alignment toward the Vitis 201

vinifera genome with perfect matches, gave 63.7% (5,580,976) for vine ZA505-1N, and 61.7% 202

(4,826,737), for vine ZA505-2A, matching reads (not shown). 203

De novo assembly of the reads by Velvet with k-mer of 15 and 17 gave 6493/2557 or 204

6254/2326 contigs from ZA505-1N and ZA505-2A libraries, respectively (Table 3). BLASTN and 205

BLASTX searches identified a similar number of contigs homologous to GPGV IT (7) and GPGV 206

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SK30 (8) in both libraries, but their relative distribution toward these virus isolates was different. 207

Particularly, a larger number of contigs from vine ZA505-2A (44 and 45 with k-mer 15 and 17, 208

respectively) was homologous to the symptomatic GPGV IT isolate, whereas contigs generated 209

from the symptomless vine ZA505-1N were largely homologous (51 and 31 with k-mer 15 and 17, 210

respectively) to the Slovak GPGV SK30 isolate. GRSPaV showed the highest number of contigs 211

(Table 3), whereas similar low number of HSVd and GYSVd-1 contigs were found in both libraries. 212

A striking difference with the previously described virome (7) was the exclusive presence of 213

GRVFV-specific contigs in vine ZA505-2A. After this initial search, a dedicated database 214

composed of representative genomes of GPGV, GRSPaV, GRVFV, HSVd and GYSVd-1 was used 215

to probe ZA505-1N and ZA505-2A libraries by SOAP, allowing two nucleotide mismatches. These 216

alignments (Supplementary Table 1) showed that a higher number of 18-26 nt reads matched the 217

GPGV genome in the symptomless ZA505-1N vine with respect to the symptomatic ZA505-2A. 218

Moreover, GPGV-homologous reads represented the majority of virus- and viroid-associated reads 219

in both vines, with the only exception of GYSVd-1 in vine ZA505-1N. As expected, GRVFV-220

homologous reads were found only in vine ZA505-2A. 221

In order to verify the possible involvement of GRVFV in disease symptoms, the previously 222

sRNA-sequenced vines (7), ZA505-1A (symptomatic) and ZA505-2N (symptomless), and the 223

currently investigated ZA505-2A and ZA505-1N plants, were tested by RT-PCR for the presence of 224

this virus. GRVFV primers selected in two different laboratories (Dr. M. Al Rwhanih and Dr. S. 225

Sabanadzovic, personal communications) failed to detect the virus, likely because of genome 226

variability and limited sequence information available.Two additional primer sets designed on 227

GRVFV sRNA assembled sequences (19) were therefore tested. The expected 705 nt (set a) and 228

1237 nt (set b) fragments were amplified from symptomatic ZA505-2A and symptomless ZA505-229

2N vines, respectively (Figure 1) but not from symptomatic ZA505-1A and symptomless ZA505-230

1N vines. An in-depth analysis of GRVFV reads showed that their number (Table 3) is negligible 231

(i.e. 24,586 18-26 redundant reads) as compared to GPGV reads. This was shown by a scarce 232

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GRVFV genome coverage (61%) and average coverage depth (13X) (Supplementary Figure 2). 233

These data suggest a limited GRVFV replication, likely as a result of RNA silencing activity in the 234

tissue analyzed. Since these findings were indicative of an inconsistent association of GRVFV with 235

symptoms, no further attention was payed to this virus. 236

Collectively, these results ruled out the involvement in disease expression of viruses or viroids 237

other than GPGV, thus supporting the hypothesis that strains of this virus with diverse biological 238

traits existed, in line with data resulting from biological indexing. 239

Genetic diversity of GPGV isolates. The genome consensus sequences of GPGV isolates 240

ZA505-2A, ZA505-1N and ZA505-2N (ZA505-2Acons, ZA505-1Ncons and ZA505-2Ncons) were 241

obtained by short read alignment toward the GPGV IT reference sequence and phylogenetic 242

relationships were investigated by nucleotide comparisons with GPGV IT and the three Slovak 243

isolates SK30, SK01 and SK13 (8). The good genome coverage (Figure 2) (average coverage depth 244

of ZA505-2Acons: 51X; ZA505-1Ncons: 70X; ZA505-2Ncons: 61X) of the three consensus 245

sequences, each covering >98% of the viral genome, made us confident that a phylogenetic 246

relationship could correctly be established. Maximum likelihood phylogeny, inferred under the 247

Hasegawa-Kishino-Yano (HKY) model of nucleotide substitution (9), grouped the isolates from 248

symptomatic vines ZA505-1A and ZA505-2A in a clade strongly supported (100% of replicates) by 249

bootstrap analysis (Figure 3). As expected, clustering of the Slovak isolates SK01, SK13 and SK30 250

is strongly supported, whereas the topology of isolate ΖΑ505-1N is not clearly defined, although its 251

belonging to the clade of symptomless isolates is certain. Results of this analysis suggest that 252

GPGV ZA505-1A and ZA505-2A isolates underwent a selection mechanism that induces symptoms 253

in grapevine. Strikingly, these phylogenetic results mirror those found studying contig homologies, 254

in that a larger number of contigs from vine ZA505-2A align to the symptomatic GPGV IT isolate 255

ZA505-1A, whereas the opposite occurs with contigs generated from the symptomless vine ZA505-256

1N, which prevalently matches the Slovak isolate SK30. To rule out the potential bias determined 257

by the reference isolate (ZA505-1A) the same analysis was performed after generating ZA505-2A, 258

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ZA505-1N and ZA505-2N consensus sequences toward the GPGV SK30 isolate. A comparable 259

clustering was obtained when phylogeny was determined with these SK30-derived consensus 260

sequences (not shown). 261

To confirm the existence of genetic differentiation in symptomatic GPGV isolates, two viral 262

genome regions were analyzed, one encompassing the 3’end of the movement protein (MP) and the 263

5’ end of the coat protein (CP) genes (MP/CP) and the other the RNA dependent RNA polymerase 264

domain (RdRp) of the replicase gene. Sequences from 45 and 20 MP/CP and RdRp gene fragments, 265

of isolates from different grapevine accessions, composed of an almost equal number of vines 266

showing symptoms or not, were selected for the analysis (Table 1). The majority of samples, 267

belonging to the cvs. Pinot gris and Traminer, was collected in different vineyards in Trentino in 268

2011-2014. Of the 45 MP/CP gene sequences, 37 and 5 were from Trentino and Apulia viral 269

isolates, respectively (18), whereas the 3 Slovak sequences were retrieved from the NCBI database 270

(8). Similarly, the group of viral strains from which the RdRp sequences were obtained, was 271

composed of 15 Trentino, 2 Apulia and 3 Slovak isolates, the latter retrieved from the NCBI 272

database. Isolates from Apulia were chosen from symptomless vines and those from the Slovak 273

Republic were also reported as symptomless (8). In order to identify the predominant viral sequence 274

in each accession from Apulia and Trentino, three different plasmids, recombinant for the MP/CP- 275

or RdRp-amplified regions were sequenced and a consensus sequence was generated, and used for 276

phylogenetic assessment. Nucleic acid sequences determined in this study are available in the 277

EMBL Nucleotide Sequence Database with accession numbers LN606702 to LN606758. No 278

recombination was found using RDP3, allowing to infer phylogenetic relationships by the 279

Maximum Likelihood method under the same substitution model as above. The tree generated using 280

MP/CP sequences clearly differentiated isolates from symptomatic grapevines which was supported 281

in 82% of bootstrap replicates (Figure 4A), whereas isolates from symptomless grapevines clustered 282

in two strongly supported clades (98 and 96% replicates). The same topology, clustering in a well 283

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supported clade (83% bootstrap replicates) all the isolates originating from symptomless vines, was 284

obtained when the RdRp domain was analysed (Figure 5). 285

Prior to the phylogenetic analysis under a Bayesian framework, the existence of a temporal 286

signal in our MP/CP sequence data was evaluated. A linear regression between the genetic distance 287

from the root and the sampling time by using Path-O-Gene (not shown), yielded a correlation 288

coefficient of 0.2635, which is weakly indicative of a linear increase of nucleotide substitution over 289

time. This very low value is plausible, considering the limited five-year time period (2010-2014) of 290

sampling. A Maximum Clade Credibility (MCC) tree was obtained using the HKY best-fitting 291

model of nucleotide substitution using the BEAST package. Phylogenetic clustering of MP/CP 292

sequences confirmed the previously observed partitioning of GPGV isolates in two lineages that 293

comprise the symptomatic and symptomless phenotypes, and this was supported by strong posterior 294

probabilities (Figure 4B). Similarly, the analysis of the RdRp region showed the same distinction, 295

with the difference that clustering of the symptomatic isolates was, in this region, split into two 296

branches, one of them containing the grapevine accessions MER(FA)1A (cv. Merlot) and 297

BE(FA59)1A (cv. Pinot noir) (Figure 5B). 298

A careful observation of the aligned MP/CP sequences from GPGV isolates, disclosed a 299

polymorphism involving the MP stop codon TAA at position 6684 of the GPGV IT genome 300

sequence. Differently from the Slovak isolates GPGV SK01, SK13 and SK30, the isolate GPGV IT 301

should have a 6 amino acid shorter MP, because of the T/C polymorphism involving this stop codon 302

(Supplementary file 1). The alignment of all the MP/CP sequences revealed that the TAA stop 303

codon is maintained in all but one isolate [ZA(PA)P2] originating from symptomatic vines, which, 304

consequently, have a 369 amino acid-long MP whereas MPs of isolates from symptomless vines are 305

375 amino acid-long. Presumably, the inconsistency of isolate ZA(PA)P2 is in line with its 306

unresolved topology (Figure 4A and B). To find out if the observed lineage distribution (i.e. 307

symptomatic vs symptomless isolates) could be exclusively related to this T/C polymorphism this 308

nucleotide was artificially changed in a set of 10 randomly chosen sequences, either from isolates 309

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showing symptoms or not. The resulting altered alignment generated the same lineage distribution 310

observed in phylogenetic analysis with real data. This test demonstrated that the T/C polymorphism 311

is not the sole responsible for lineage distinction, but likely, is part of a more general GPGV 312

phylodynamic behavior. 313

Field survey and virus frequency. Field surveys were done in several vineyards of Val 314

d’Adige and Vallagarina for five consecutive seasons (2010 to 2014), which disclosed the presence 315

of symptoms also on cv. Merlot (Table 1). Collection from symptomless vines in the same vineyard 316

showed that 75 of 92 (82%) vines were infected with GPGV (Table 4). In this viticultural area, 317

virus association with symptoms was found in 59 of 75 GPGV-infected vines (79%). Conversely, 318

17 symptomless vines were negative for GPGV in RT-PCR. All tested vines were negative for 319

viruses associated with grapevine leafroll, rugose wood and infectious degeneration, but were 320

occasionally infected by GRSPaV or GFkV, with no consistent association with the disease (Table 321

1). 322

323

DISCUSSION 324

A series of common findings connotes this new grapevine disease to which GPGV is associated 325

in all viticultural areas of occurrence: (i) symptoms were first observed in 2001 in Slovenia (20) and 326

2003 in Trentino (7); (ii) the disease seems to spread and cause economic losses particularly in 327

premium wine cultivars (20, 22); (iii) wherever the disease occurs no relevant grapevine viruses are 328

associated with it, except for GPGV (7, 20, http://archives.eppo.int/EPPOReporting/2014/Rse-329

1401.pdf). 330

GPGV was also found in symptomless grapevines (7, 8, 331

http://archives.eppo.int/EPPOReporting/2014/Rse-1401.pdf), raising doubts on its involvement in 332

the disease, a behavior already reported for the close relative GINV (10). The disease emergence 333

and spread support the recent occurrence of a new pathogen correlated with it, that experimental 334

evidence associates with the presence of GPGV, at least in the viticultural areas of Trentino. This 335

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conclusion is supported by the consistent association of GPGV with symptomatic but not 336

symptomless vines, and more strongly, by the results of indexing that disclosed the existence of 337

severe strains of the virus, pathogenic to Pinot gris and Traminer but not to the indicators Cabernet 338

franc and Vitis rupestris. Both findings, together with multiple infections, likely explain the lack of 339

association of GPGV with symptoms in Czech and Slovak vines (8). 340

NGS investigations further defined and confirmed the already known virome (7) and, besides 341

excluding the existence of unknown viruses or viroids, allowed to re-assemble the almost complete 342

genomic RNAs of three additional GPGV isolates from vines ZA505-2A, ZA505-1N and ZA505-343

2N. Phylogenetic comparisons of available complete or consensus genomic RNAs delineate a 344

“speciation” of symptomatic isolates ZA505-1A and ZA505-2A, whereas symptomless isolates 345

from Trentino are grouped in a broader clade, together with those reported from the Slovak 346

Republic. This observed GPGV evolutionary dynamic, indicating the existence of virulent and 347

latent variants, was also confirmed by studies, on a larger number of isolates, in the MP/CP and 348

RdRp genomic regions, in which two distinct genetic lineages, grouping, respectively, symptomless 349

and symptomatic vines, were identified. Polymorphism in the MP stop codon confirms the GPGV 350

tendency, already observed by Glasa et al. (8), to undergo an unusual nucleotide divergence and 351

suggests a biological significance of this finding. The present investigations, in which the stop 352

codon polymorphism was artificially modified, did not change the resulting topology, suggesting 353

that it is part of a GPGV general evolutionary trend and not the sole responsible for symptom 354

association. 355

All these findings point towards the emergence of a new plant virus that underwent a recent 356

evolution, whose origin is still obscure and will require further studies involving a larger number of 357

samples. Factors that favor new virus disease emergence have been associated with ecological 358

change or intensive agronomical practices (5), both common and frequent events occurring in some 359

premium viticultural areas where GPGV was found. One of these factors consists in the recurrent 360

renewal of vineyards with new cultivars to support market requests. Due to the continuous report of 361

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new GPGV detections in Italy and other European countries, a collaborative effort is requested to 362

clarify its biology and epidemiology, taking into account that GINV, the other known grapevine 363

trichovirus, is transmitted by mites. 364

365

ACKNOWLEDGMENTS 366

367

The research was supported by the Fondazione Edmund Mach, San Michele all’Adige (Trento), 368

Italy, in the frame of the Project ”Studio di una nuova malattia della vite in Trentino”. A. 369

Giampetruzzi was supported by a fellowship of the National Research Council, Project “CISIA - 370

Valorizzazione delle risorse genetiche di colture mediterranee attraverso approcci di metagenomica, 371

trascrittomica e analisi funzionale per la caratterizzazione di germoplasma autoctono, endofiti, 372

agenti di biocontrollo e fitopatogeni (METAGERM)”. The authors wish to thank Prof. G.P. Martelli 373

for critically reading and revising the manuscript. 374

375

376

377

378

379

380

381

382

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384

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473

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TABLE 1. Grapevine samples analyzed in the present study. GPGV infection and presence of 493

symptoms are indicated (+); (-) indicates no symptoms or absence of GPGV. Additional viruses 494

detected in each sample are reported. The “Sequence” column specifies the isolates used for cloning 495

and sequencing the corresponding MP/CP and RdRp sequences. 496

497

Cultivar Place of

samplingd

Sample id. GPGV Symptoms Sequence Viruses Year of

sampling

Pinot gris Zablani ZA505-1A + + RdRp, MP/CP

GRSPaV,

GSyV-1, HSVd,

GYSVd-1

2010

Pinot gris Zablani ZA505-2A + + RdRp,

MP/CP

GRSPaV,

GRVFV,

HSVd,

GYSVd-1

2013

Pinot gris Zablani ZA505-1N + - RdRp, MP/CP

GRSPaV,

HSVd,

GYSVd-1

2013

Pinot gris Zablani ZA505-2N + - RdRp,

MP/CP

GRSPaV,

GRVFV,

GSyV-1,

HSVd, GYSVd-1

2010

Traminer Filippi FI5A + +

GRSPaV 2013

Traminer Filippi FI5N - -

GRSPaV,

GFkV 2013

Pinot gris S.Donà SD7-3 - 6A + +

GRSPaV 2013

Pinot gris S.Donà SD8 - 8-9A + +

GRSPaV,

GFkV 2013

Traminer Navesel NAV9161N - -

GRSPaV 2013

Traminer Navesel NAV9181N + - RdRp,

MP/CP GRSPaV 2013

Pinot gris Navesel NAV5051N + -

GRSPaV 2013

Pinot gris Navesel NAV5141N - -

GRSPaV 2013

Pinot noir Navesel NAV1911N - -

GRSPaV 2013

Pinot noir Navesel NAV1851N + +

GRSPaV,

GLRaV-3 2013

Pinot noir Navesel NAV2011N + -

GRSPaV,

GLRaV-3 2013

Pinot gris Paoli PA505v.2N + +

GRSPaV 2013

Pinot gris Paoli PAv514v.2

N + +

GRSPaV 2013

Pinot gris Zablani ZA505-4N + -

GRSPaV 2013

Pinot gris Zablani ZA505-3N + - RdRp,

MP/CP GRSPaV 2013

Pinot gris Zablani ZA505-10N + + MP/CP GRSPaV 2013

Pinot gris S.Donà SD4 -4 -6A + +

GRSPaV 2013

Pinot gris S.Donà SD2-12-4 A + +

GRSPaV 2013

Traminer Navesel NAV916-

2N - -

GRSPaV 2013


3N - -

GRSPaV 2013

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2N - -

GRSPaV 2013


3N + +

GRSPaV 2013

Pinot gris Navesel NAV505-

2N - -

GRSPaV 2013

Pinot gris Navesel NAV514-

2N - -

GRSPaV 2013

Pinot noir Navesel NAV191-

2N + +

GRSPaV, GFkV

2013


2N + +

GRSPaV,

GFkV 2013


2N - -

GRSPaV 2013

Pinot gris Zablani ZA505-3A + + RdRp GRSPaV,

GFkV 2011

Pinot gris Zablani ZA505-4A + +

GFkV,

GRSPaV 2013


MP/CP GRSPaV 2011

Pinot gris Zablani ZA505-6A + + MP/CP GRSPaV 2013

Pinot gris Zablani ZA505-7A + + MP/CP GRSPaV 2013


MP/CP GRSPaV 2013

Pinot gris Zablani ZA505-9Aa + + RdRp,

MP/CP GRSPaV 2011, 2013

Pinot gris Zablani ZA505-5N + -

GRSPaV,

GFkV 2013

Pinot gris Zablani ZA505-6N + - MP/CP GRSPaV 2013





GFkV, GRSPaV

2013


GRSPaV 2013

Traminer Filippi FI3A + + MP/CP GFkV,

GRSPaV 2013

Traminer Filippi FI4A + + MP/CP GRSPaV 2013

Traminer Filippi FI6AV + + MP/CP GRSPaV 2013

Traminer Filippi FI7AV + + MP/CP GFkV 2013

Traminer Filippi FI8AV + + MP/CP - 2013

Traminer Filippi FI1N - -

GFkV, GRSPaV

2013

Traminer Filippi FI2N + -

- 2013

Traminer Filippi FI3N + - GRSPaV 2013

Traminer Filippi FI4N + - MP/CP GRSPaV 2013

Traminer Filippi FI6NV - - - 2013

Traminer Filippi FI7NV - -

GFkV,

GRSPaV 2013

Traminer Filippi FI8NV - -

- 2013

Traminer Filippi FI9NV + +

GFkV, GRSPaV

2013

Vitis riparia

Screenhouse VASO C - -

GRSPaV 2012

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Traminer Screenhouse 916VASO

A - -

GRSPaV 2012

Traminer Screenhouse 918 VASO

A + +

GRSPaV 2012

Pinot gris Screenhouse 505 VASO

A - -

GRSPaV 2012

Pinot gris Camp/cpo 513 VM 5N + +

GRSPaV 2012

Pinot gris Zablani

(Pancher) ZA(PA)P1 + + MP/CP - 2013

Pinot gris Zablani


Pinot gris Zablani

(Pancher) ZA(PA)P3 + +

- 2013

Pinot gris Zablani


Pinot gris Zablani

(Pancher) ZA(PA)P5 + +

GRSPaV 2013

Pinot gris

Cadino

(Faedo

Endrizzi)

CAD(FE)P1 + +

GRSPaV 2013

Pinot gris

Cadino

(Faedo

Endrizzi)

CAD(FE)P2 + +

GRSPaV,

GFkV 2013

Pinot gris

Cadino

(Faedo Endrizzi)

CAD(FE)P3 + +

GRSPaV 2013

Pinot gris

Cadino

(Faedo

Endrizzi)

CAD(FE)P4 + +

GRSPaV 2013

Pinot gris

Cadino

(Faedo

Endrizzi)

CAD(FE)P5 + +

GFkV 2013

Pinot gris Lavis

(Gottardi) LA(GO)P1 + +

GRSPaV 2013

Pinot gris Lavis


GRSPaV 2013

Pinot gris Lavis


- 2013

Pinot gris Lavis


GRSPaV, GFkV

2013

Pinot gris Lavis

(Gottardi) LA(GO)P5 + + MP/CP GFkV 2013

Traminer Bellaveder

(Faedo) BE(FA)P1 + +

GVA 2013

Traminer Bellaveder

(Faedo) BE(FA)P2 + + MP/CP - 2013

Traminer Bellaveder


- 2013

Traminer Bellaveder

(Faedo) BE(FA)P4 + + MP/CP - 2013

Traminer Bellaveder


GRSPaV 2013

Pinot gris Zablani ZA6Ncloros

i + -

RdRp,

MP/CP GRSPaV 2011

Pinot noir Bellaveder BE(FA)1A + + GRSPaV 2011

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(Faedo)

Merlot Bellaveder

(Faedo)

MER(FA)1

A + +

RdRp,

MP/CP GRSPaV 2011

Pinot noir Bellaveder

(Faedo)

BE(FA59)1

A + +

RdRp,

MP/CP GRSPaV 2011

Traminer Bellaveder

(Faedo) BE(ac) + + MP/CP GRSPaV 2011

Traminer Bellaveder

(Faedo) BE5A + + MP/CP GRSPaV 2011

Traminer Ala acari

Pozzo basso ALA-P4 + + MP/CP GRSPaV 2014

Pinot noir Bellaveder PN-SO41A + + MP/CP GRSPaV,

GFkV 2011

Pinot gris Screenhouse CLONE505

3Tb + + MP/CP

GRSPaV,

GRVFV, GSyV-1,

HSVd,

GYSVd-1

2014

Supernov

a Mola di Bari MOLA14 + - MP/CP

GRSPaV,

GFLV, 2014

Supernov

a Mola di Bari MOLA10 + -

RdRp,

MP/CP

GRSPaV,

GFkV, GFLV 2014

Black

magic Mola di Bari MOLA1 + -

RdRp,

MP/CP GRSPaV 2014

Supernov

a Mola di Bari MOLA6 + -

RdRp,

MP/CP

GRSPaV,

GFkV, GFLV 2014

Supernov

a Mola di Bari MOLA3x3 + -

RdRp,

MP/CP

GRSPaV,

GFkV, 2014

unknown Cachtice,

Slovakia SK01 + -

RdRp,

MP/CP Mult. Inf.c 2012

unknown Cachtice,

Slovakia SK13 + -

RdRp,

MP/CP Mult. Inf

c. 2012

Veltliner Pezinok, Slovakia

SK30 + - RdRp, MP/CP

GFkV,

ArMV,

GLRaV-1

2012

498 are-tested in 2011 and 2013 only for MP/CP 499 b505 VASO A grafted on ZA505-1A 500

cMultiple infection 501 dZablani, Filippi, Navesel, Paoli, Bellaveder, S. Donà, Camp/cpo indicate vineyard locations; 502

Cadino, Ala, Lavis, Mola di Bari, Cachtice and Pezinok indicate towns; Screenhouse indicates 503

screenhouse-maintained vines 504

505

506

507

508

509

510

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511

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TABLE 2. Summary of the assays of biological indexing. Bud grafted plants were scored for symptoms during four years but only two observations 512

(2010 and 2014) are reported. Green grafted plants were produced and observed only during 2014. 513

514 W Rootstock vines: ZA505-1A, ZA505-2A, ZA505-3A, ZA505-4A, ZA505-5A, ZA505-7A, ZA505-8Aand ZA505-9A. 515

X Rootstock vines: ZA505-1N, ZA505-2N, ZA505-3N, ZA505-4N, ZA505-5N, ZA505-6N, ZA505-7N, ZA505-8N and ZA505-9N.

516

Y Rootstock vines: ZA505-1A, ZA505-2A.

517

Z Rootstock vines: ZA505-1N, ZA505-2N. 518

NA: Not applicable

519

520

Rootstock Indicator No. of plants No. plants with symptoms No. of plants No. plants with symptoms

2010 2014

Bud grafting

P. gris with symptomsw

Cabernet f. 4 0 3 0

V. rupestris 4 0 1 0

P. gris 16 12 13 13

Traminer 3 3 1 1

P. gris without symptomsx

Cabernet 6 0 5 0

V. rupestris 8 0 2 0

P. gris 25 0 20 0

Traminer 4 0 4 0

Green

grafting P. gris with symptoms

y

P. gris NA NA 3 2

Traminer NA NA 8 8

P. gris without symptomsz Traminer NA NA 3 0

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TABLE 3. Distribution of contigs with virus and viroid homologies in the sRNA libraries 521

ZA505-1N and ZA505-2A. De novo-assembling of short reads was performed by Velvet 522

using k-mer 15 and 17. Homologies were assessed by BLASTN or BLASTX searches toward 523

a NCBI virus and viroid database. Numbers of contigs homologous to the listed viruses and 524

viroids and corresponding N50 values are indicated. 525

526

527

Grapevine accession ZA505-1N ZA505-2A

k-mer 15 17 15 17

total number of contigs(N50) 6493 2557 6254 2326

Grapevine Pinot gris virus IT

Grapevine Pinot gris virus SK30

Grapevine rupestris vein feathering virus

Grapevine rupestris stem pitting associated virus

Grapevine yellow speckle viroid 1

Hop stunt viroid

9(77)

51(64)

0(0)

295(63)

11(29)

9(38)

6(176)

31(105)

0(0)

141(71)

22(33)

9(58)

44(121)

23(63)

70(80)

223(86)

9(51)

8(61)

45(120)

11(92)

57(78)

119(88)

3(186)

3(145)

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

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548

TABLE 4. Survey of GPGV association with symptoms in Trentino by RT-PCR. Presence or 549

absence of the virus are indicated by (+) and (-), respectively. 550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585

586

587

588

589

Vines with symptoms N (%) Symptomless vines N (%) Tot

GPGV + 59 (79) 16 (21) 75

GPGV - 0 (0) 17 (100) 17

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CAPTIONS TO FIGURES 590

Figure 1. GRVFV detection by RT-PCR. Amplicons obtained from ZA505-1A (1A), ZA505-2A 591

(2A), ZA505-1N (1N) and ZA505-2N (2N) vines using two sets of primers (a) and (b) are separated 592

by gel electrophoresis. DNA molecular weight marker and PCR control without cDNAs are 593

indicated by (Mk) and (C), respectively. 594

595

Figure 2. Graph plots showing the number of 20-24 unique viral sRNAs of ZA505-1N, ZA505-2A 596

and ZA505-2N distribution along the GPGV IT RNA. GPGV genome organization is depicted 597

below the plots. Sense and antisense reads are collapsed above the x-axis, reporting the nucleotide 598

numbering, whereas the y-axis indicates nucleotide coverage. Genome covered bases and average 599

coverage depth are reported. 600

601

Figure 3. Maximum likelihood tree inferred with available full-length or GPGV genome consensus 602

sequences. The tree was generated under the HKY + Γ model of nucleotide substitution. Branches 603

supported by a minimum of 50% of 1000 bootstrap replicates are indicated. Scale indicates units in 604

nucleotide substitutions per site. 605

606

Figure 4. Maximum likelihood (A) and Bayesian (B) phylogenetic trees inferred with 45 GPGV 607

MP/CP gene sequences. Trees were generated under the HKY + Γ model of nucleotide substitution. 608

Branches supported by a minimum of 50% of 1000 bootstrap replicates (A) and node significances 609

by Bayesian posterior probabilities (B) are indicated. Branches are condensed. The grey and empty 610

shadowing group isolates from symptomatic or symptomless vines, respectively. 611

612

Figure 5. Maximum likelihood (A) and Bayesian (B) phylogenetic trees inferred with 20 GPGV 613

RdRp sequences. Trees were generated under the HKY + Γ model of nucleotide substitution. 614

Branches supported by a minimum of 50% of 1000 bootstrap replicates (A) and node significances 615

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by Bayesian posterior probabilities (B) are indicated. Branches are condensed. The grey and empty 616

shadowing group isolates from symptomatic or symptomless vines, respectively. 617

618

Supplementary Figure 1. Size distribution of redundant or unique small RNAs in libraries prepared 619

from ZA505-1N (symptomless) and ZA505-2A (symptomatic) vines. Reads numbers and sizes are 620

indicated in the y and x axes, respectively. 621

622

Supplementary Figure 2. Graph plots showing the number of 20-24 unique viral small RNAs of 623

ZA505-2A distribution along the GRVFV genome. Sense and antisense reads are collapsed above 624

the x-axis, reporting the nucleotide numbering, whereas the y-axis indicates nucleotide coverage. 625

Genome covered bases and average coverage depth are reported. 626

627

Supplementary file 1. Alignment of 45 MP/CP nucleotide sequences used for inferring phylogeny. 628

Dots indicate identical nucleotides. Sequences from symptomatic and symptomless vines have red 629

and black fonts, respectively. The 6684 T/CAA and in frame 6702 TGA stop codons are highlighted 630

in yellow. The T/C polymorphism in position 6684 generates a shorter MP in all symptomatic 631

isolates, with the exception of ZA(PA)PA2. 632

633

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37x26mm (300 x 300 DPI)

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75x64mm (300 x 300 DPI)

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50x28mm (300 x 300 DPI)

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72x59mm (300 x 300 DPI)

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45x23mm (300 x 300 DPI)

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80x72mm (300 x 300 DPI)

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SUPPLEMENTARY TABLE 1. Viral and viroid sRNAs constituting the virome of ZA505-1N and ZA505-2A as obtained by SOAP alignments to the

corresponding reference genomes with a tolerance of two mismatches. The 18-26 nt range of sizes and selected 21 and 24 nt long molecules are

showed.

ZA505-1N ZA505-2A

18-26 nt reads 21 nt 24 nt 18-26 nt reads 21 nt 24 nt

unique redundant unique redundant unique redundant unique redundant unique redundant unique redundant

Grapevine Pinot gris virus IT 23.668 195.578 9.205 127.520 1.075 2.391 17.437 104.482 7.575 71.306 645 1.034

Grapevine rupestris vein feathering virus 0 0 0 0 0 0 4.130 24.586 2.323 19.597 25 28

Grapevine rupestris stem pitting associated virus 17.938 66.260 7.851 40.769 530 717 5.639 18.596 2.604 11.819 121 146

Grapevine yellow speckle viroid 1 5.455 195.778 1.468 129.562 1.119 33.080 3.723 53.590 925 24.116 832 19.098

Hop stunt viroid 3.097 43.453 715 17.757 641 12.967 3.597 47.613 803 19.020 740 14.496

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MOLA10 A A C A T T G A T A G G T A C T T T T C A C T G G T G C C T T C G G A C A G T T C A T C C A T T G T T A A A T C T T T T G T T G A C A G T T A C A A A A G A [ 78]

MOLA6 . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

MOLA3x3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

MOLA14 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

MOLA1 . . . . . . . . . . A . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . A C A . . . . . . [ 78]

SK30 C . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

SK13 C . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

SK01 C . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

Clone505 3T . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-1A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

FI8AV . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

FI7AV . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

FI6AV . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

FI4N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

FI4A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . T . . . . . . . . . . . . [ 78]

FI3A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

BE5A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

BE(FA)P4 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

BE(FA)P2 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

BE(ac) . . . . . . . . . . . . . . . . . . . . . . . . . . A . . C . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

NAV9181N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-9A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-5A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

PN-SO41A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-3N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

BE(FA59)1A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-2N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-2A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-1N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . T . . . . . . . . . . . . [ 78]

ZA(PA)P4 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA(PA)P2 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA(PA)P1 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-10N . . . . . . . . C . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . G [ 78]

ZA505-9A 2013 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-8N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-7N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-7A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-6N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-6A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

LA(GO)P5 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

MER(FA)1A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ALA-P4 . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-9N . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-8A . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

ZA505-6Nclor . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [ 78]

MOLA10 A A A G G T C T G T T T A G C C T A A A A T C T A A T G T C A G G T T A C A C A A A A A T G A T G T T G G G C C C T T G T C T T T T A A A G G A A A C A G T [156]

MOLA6 . . . . . . . . . . . . . R . . . . . . . . . . G . . . . . . . . . . R . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

MOLA3x3 . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

MOLA14 . . . . . . . . . . . . . A . . . . . . . . . . G . . . . . . . . . . G . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

MOLA1 . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . [156]

SK30 C . . . . . . . . . . . . . . . . . . . . G . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

SK13 C . . . . . . . . . . . C . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

SK01 C . . . . . . . . . . . . . A . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

Clone505 3T . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-1A . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . G . . . . [156]

FI8AV . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

FI7AV . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

FI6AV . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

FI4N . . . . . . . . . . . . . A . . . . . . . . . . G . . . . . . . . . . G . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

FI4A . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . [156]

FI3A . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

BE5A . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

BE(FA)P4 . . . . . . . . . . . . . A . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

BE(FA)P2 . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

BE(ac) . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

NAV9181N . . . . . . T . . . . . . . T . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-9A . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-5A . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

PN-SO41A . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-3N . . . . . . . . . . . . . A . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

BE(FA59)1A . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-2N . . . . . . . . . . . . . A . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . C . . G . . . . . . . . . [156]

ZA505-2A . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-1N . . . . . . . . . . . . . A . . . . . . . . . . G . . . . . . . A . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA(PA)P4 . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA(PA)P2 . . . . . . . . . . . . . . . . . G . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . [156]

ZA(PA)P1 . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-10N . G . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-9A 2013 . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-8N . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-7N . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-7A . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-6N . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-6A . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

LA(GO)P5 . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

MER(FA)1A . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ALA-P4 . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-9N . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

ZA505-8A . . . . . . . . . . . . . C . . . . . . . . . . G . . . . . . . . . . . . . . . G G . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . [156]

ZA505-6Nclor . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [156]

MOLA10 C T T T T C T T T C A G G A A A G C A C A T C A A A T A A A G A A A T G G C T C C T G T T C G C A C T G A A A G T G A A T C A T G T C T A A A T G T T T T C [234]

MOLA6 . . . . . . . . . . . . . . . . . T G . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . G . . . . . . . . . . . . . . . [234]

MOLA3x3 . . . . . . . . . A . . . . . . . T . . . . . G . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

MOLA14 . . . . . . . C . . . . . . . G . . G . . . . . . . C . . . . . . . . . . . C . . . . . . . . A . . . . . . . A . . . . . . . . . . . . G . . . . . . . . . [234]

MOLA1 . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . G . . C . . . . . . [234]

SK30 C . . . . . . . . . . . . . . . . . T . . . . . G . . . . . . . . . . . . . . C . . . . . . . . T . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

SK13 C . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . T . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

SK01 C . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . C . . . T . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

Clone505 3T . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-1A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

FI8AV . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . G A . . . . . . . . . . . C . . . . . . . . . . [234]

FI7AV . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

FI6AV . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

FI4N . . . . . . . C . . . . . . . G . . G . . . . . . . C . . . . . . . . . . . C . . . . . . . . A . . . . . . . A . . . . . . . . . . . . G . . . . . . . . . [234]

FI4A . . . . . . C . . . . . . . . G . T . . . . . . . . C . . . . . . . . . . . C . . . . . . . A . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . [234]

FI3A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

BE5A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

BE(FA)P4 . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

BE(FA)P2 . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

BE(ac) . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

NAV9181N . . . . . T C . . . . . . . . . . T . . . . . . . . . . . . . . . . C . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

ZA505-9A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-5A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

PN-SO41A . . . . . . C . . . . . . . . G . T . . . . . G . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-3N . . . . . . . C . . . . . . G G . T . . . . . . . . C . . . . . . . . . . . C . . . . . . . . A . . . . . . . A . . . . . . . . . . T . . . . . . . . . . . [234]

BE(FA59)1A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-2N . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

ZA505-2A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A A . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-1N . . . . . . . C . . . . . . . G . T . . . . . . . . C . . . . . . . . . . . . . . A . . . . . A G . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

ZA(PA)P4 . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA(PA)P2 . . . . . . . . . . . . . . . G A T . . . . . . . . . . . . . . . . . . . . C . . . . . . . A . . . . . . . . A . . . . . . . . . . . C G . . . . . . . . . [234]

ZA(PA)P1 . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . G . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-10N . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-9A 2013 . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-8N . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

ZA505-7N . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

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ZA505-7A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . G . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-6N . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

ZA505-6A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

LA(GO)P5 . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

MER(FA)1A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ALA-P4 . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-9N . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

ZA505-8A . . . . . . C . . . . . . . . G . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . C . . . . . . . . . . [234]

ZA505-6Nclor . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . [234]

MOLA10 T C C A A A G G T T C T G G T G A T C C A A T G G T A A A G A T G G T T A A G T C T A A A T C T G G C T G T G C T G A A A A T A G T G C T G A A T T T G A A [312]

MOLA6 . . . . . G . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . [312]

MOLA3x3 . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

MOLA14 . . . . . G . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

MOLA1 . . . . . G . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

SK30 C . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

SK13 C . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

SK01 C . . . . G G . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

Clone505 3T . . A . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-1A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

FI8AV . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

FI7AV . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

FI6AV . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

FI4N . . . . . G . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

FI4A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

FI3A . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

BE5A . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

BE(FA)P4 . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

BE(FA)P2 . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

BE(ac) . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

NAV9181N . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-9A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-5A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

PN-SO41A . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-3N . . . . . G . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

BE(FA59)1A . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-2N . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-2A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-1N . . . . . G . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA(PA)P4 . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA(PA)P2 . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA(PA)P1 . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-10N . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-9A 2013 . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-8N . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-7N . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-7A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-6N . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-6A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

LA(GO)P5 . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

MER(FA)1A . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ALA-P4 . . . . . G . . . . T . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-9N . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-8A . . . . . G . . . . . . . . G . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

ZA505-6Nclor . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [312]

MOLA10 A G G T A T C T A A A G C A A G A T T A C A A T T T T G G A A G A T A T G T C A A T T C G T C A G G A G T T G A G A T C A A C A G T C A G G A G A G A G C T [390]

MOLA6 . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

MOLA3x3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

MOLA14 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

MOLA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

SK30 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

SK13 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

SK01 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . [390]

Clone505 3T . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-1A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

FI8AV . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

FI7AV . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

FI6AV . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

FI4N . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

FI4A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . G . . . . . [390]

FI3A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

BE5A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

BE(FA)P4 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

BE(FA)P2 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

BE(ac) . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

NAV9181N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-9A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-5A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

PN-SO41A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-3N . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

BE(FA59)1A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-2N . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-2A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-1N . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA(PA)P4 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA(PA)P2 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . C T . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA(PA)P1 . . A . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-10N . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-9A 2013 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-8N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-7N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-7A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-6N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-6A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

LA(GO)P5 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

MER(FA)1A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ALA-P4 . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-9N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-8A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . [390]

ZA505-6Nclor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . [390]

MOLA10 G A T C G C G A A G C T G A G C G A G G C G A A T C A A G T A C T T C A T G G G T T G A C A G A A G G C A A C A A A A A T C T G G T C C T T G A C C A C A T [468]

MOLA6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

MOLA3x3 . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

MOLA14 . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

MOLA1 . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . T . . . . . [468]

SK30 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A A . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

SK13 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

SK01 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

Clone505 3T . . . . . . A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-1A . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . C . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

FI8AV . . . . . . A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

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FI7AV . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . Y . . [468]

FI6AV . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

FI4N . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

FI4A . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . T . . . G . . . . . . . T . . . . . . . . . . . [468]

FI3A . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

BE5A . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

BE(FA)P4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

BE(FA)P2 . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

BE(ac) . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

NAV9181N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . T . . . . . . . . . . . [468]

ZA505-9A . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T G . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-5A . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

PN-SO41A . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-3N . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

BE(FA59)1A . . . . . . A . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-2N . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-2A . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-1N . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA(PA)P4 . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA(PA)P2 . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA(PA)P1 . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . A . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-10N . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-9A 2013 . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-8N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-7N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-7A . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . A . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-6N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-6A . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

LA(GO)P5 . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

MER(FA)1A . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ALA-P4 . . . . . . A . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . C . . . . G . . . . . T . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-9N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

ZA505-8A . . . . . . A . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . T . . . . . . G . . . . . . . T . . . . . . . . . . . [468]

ZA505-6Nclor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . [468]

MOLA10 C T T C G C A A A T A T A G C A G T T G A A G G G A C C T C A G G G G A G A C A A T T T A C C C C A C C A C A A T G G T G A A G T G T C A T G A A G C T T T [546]

MOLA6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

MOLA3x3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

MOLA14 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

MOLA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

SK30 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

SK13 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

SK01 C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

Clone505 3T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-1A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

FI8AV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

FI7AV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

FI6AV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

FI4N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

FI4A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

FI3A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

BE5A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

BE(FA)P4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

BE(FA)P2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

BE(ac) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

NAV9181N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

ZA505-9A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-5A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

PN-SO41A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-3N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

BE(FA59)1A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-2N . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

ZA505-2A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-1N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA(PA)P4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA(PA)P2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA(PA)P1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-10N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-9A 2013 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-8N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

ZA505-7N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

ZA505-7A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-6N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

ZA505-6A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

LA(GO)P5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

MER(FA)1A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ALA-P4 . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-9N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

ZA505-8A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . T . . . . . T . . . . [546]

ZA505-6Nclor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . [546]

MOLA10 T A A [549]

MOLA6 Y . . [549]

MOLA3x3 . . . [549]

MOLA14 . . . [549]

MOLA1 . . . [549]

SK30 C . . . [549]

SK13 C . . . [549]

SK01 C . . . [549]

Clone505 3T . . . [549]

ZA505-1A . . . [549]

FI8AV . . . [549]

FI7AV . . . [549]

FI6AV . . . [549]

FI4N . . . [549]

FI4A . . . [549]

FI3A . . . [549]

BE5A . . . [549]

BE(FA)P4 . . . [549]

BE(FA)P2 . . . [549]

BE(ac) . . . [549]

NAV9181N . . . [549]

ZA505-9A . . . [549]

ZA505-5A . . . [549]

PN-SO41A . . . [549]

ZA505-3N C . . [549]

BE(FA59)1A . . . [549]

ZA505-2N . . . [549]

ZA505-2A . . . [549]

ZA505-1N . . . [549]

ZA(PA)P4 . . . [549]

ZA(PA)P2 . . . [549]

ZA(PA)P1 . . . [549]

ZA505-10N . . . [549]

ZA505-9A 2013 . . . [549]

ZA505-8N . . . [549]

ZA505-7N . . . [549]

ZA505-7A . . . [549]

ZA505-6N . . . [549]

ZA505-6A . . . [549]

LA(GO)P5 . . . [549]

MER(FA)1A . . . [549]

ALA-P4 . . . [549]

ZA505-9N . . . [549]

ZA505-8A . . . [549]

ZA505-6Nclor . . . [549]

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Genetic variability of Grapevine Pinot gris virus and its association with grapevine leaf mottling and deformation