BACKGROUND
in the last few years, single nucleotide polymorphisms  have become the most popular genetic marker system in both animals and plants.
their extraordinary abundance discovered in several genome sequencing projects  <cit> , combined with recent technological improvements, makes snp markers attractive for high-throughput use in marker-assisted breeding, est mapping and the integration of genetic and physical maps.
at present several snp identification methods are available such as resequencing of pcr amplicons with or without pre-screening, electronic snp  discovery in expressed sequence tag  and shotgun genomic libraries.
in these latter cases, sequences may be computationally screened for polymorphisms to distinguish true polymorphisms from sequencing errors if sufficient redundancy is present  <cit> .
unlike the first generation molecular markers, such as rflps  and rapds , snps can be detected through non-gel-based high-throughput assays, saving both time and money  <cit> .
several snp assay technologies have been developed based on various methods of allelic discrimination and detection platforms.
allele-specific hybridization, primer extension, oligonucleotide ligation and invasive cleavage represent four principal allelic discrimination reactions that can be coupled with several detection methods such as fluorescence, luminescence and mass measurements .
recently, significant efforts towards large-scale snp characterisation have been attempted in animals and plants with beadarray technology  and the snplex™ genotyping system .
the selection of an appropriate genotyping method depends on many factors including cost, potential for multiplexing and throughput, equipment, and difficulty of assay development.
until recently, only a few low- and mid-throughput methods have been used for snp discovery and genotyping in grapevine   <cit> .
the sequencing of the highly heterozygous genome of pinot noir  <cit> , clone entav  <dig>  made it possible to identify millions of esnps as a potential source for high-throughput genotyping methods.
herein we report a successful application of the snplex genotyping system, which provided  <dig> new snp-based markers anchoring the grapevine genome for future applied research programs.
RESULTS
snplex and data analysis on genomic dna
of  <dig> candidate esnps selected from non-repetitive genome contigs,  <dig> passed the design rules of the snplex assay-design pipeline and were tested in  <dig> f <dig> progeny of syrah × pinot noir  cross and in the two parental genotypes.
<dig> esnps passed the quality value using the rule-based method, with a mean of  <dig> failed snps and a 98% call rate per snpset, while the remaining  <dig> were discarded from further analyses .
of the  <dig> esnps,  <dig> were false positives.
of the remaining  <dig> esnps ,  <dig> followed the 1: <dig> or 1:2: <dig> mendelian segregation ratio based on the chi-square test, whereas  <dig> showed an unexpected segregation ratio.
within the latter class, there were  <dig> cases where one parent was heterozygous and three clusters were observed  and  <dig> cases where both parents were heterozygous and four clusters were detected .
snplex and data analysis on whole genome amplification dna
a total of  <dig> esnps combined in three 48-plex snpsets  were also tested on wga-dna of the same  <dig> individuals.
in this set,  <dig> esnps that did not pass the quality value on gdna analysis were recovered on the wga-dna test, whereas three esnps that passed the quality value on gdna failed on wga-dna .
thirteen esnps failed both gdna and wga-dna tests.
the genotyping data were thoroughly consistent between the two analyses.
resequencing analysis
to validate the data obtained with the snplex assay, six regions containing snp <dig>  snp <dig>  snp <dig>  snp <dig>  snp <dig> and snp <dig> were resequenced in syrah, pinot noir and six progeny.
four of them, snp <dig>  snp <dig>  snp <dig> and snp <dig>  showed a mendelian segregation and resequencing the corresponding regions confirmed the data obtained with the snplex analysis.
for snp <dig> and snp <dig>  which presented an additional homozygous cluster, an unexpected snp was found within  <dig> bp from the target snp in syrah and in the progeny belonging to the additional homozygous cluster .
discussion
in this work, we report the first snplex genotyping system application in higher plants, which allowed the development of markers anchoring the grapevine genome.
to date, a few low- and mid-throughput methods based on sscp and minisequencing assay have been used for snp genotyping in grape  <cit> .
snp markers have also been developed based on esnp discovery in a  <dig> × shotgun sequencing coverage of grapevine genome  <cit> .
the efficiency rate of 69% with an average call rate of 98% exceeded the level recently achieved by resequencing selected ests  and bac-end sequences  in previous study  <cit> .
although the efficiency was greatly enhanced, we observed that there were 10% systematic failed assays already detected in other snplex genotyping study  and 21% false polymorphisms.
this unexpectedly high rate of false polymorphisms might be due to the snplex probe design based on a preliminary stage of contig assembly where the coverage of each haplotype in some regions was not sufficient.
in the wga-dna pilot study the genotyping data obtained from the gdna and wga-dna analyses were thoroughly consistent, confirming previous snp genotyping studies based on beadarray  <cit>  and affimetrix technologies  <cit> .
moreover, the number of systematic failed assays was greatly reduced, from 10% to 2%, enhancing the average efficiency rate from 69% to 80%.
these results were expected since mda methods  <cit>  provide a large amount of pure dna with a uniform concentration among samples  <cit> , meeting two basic requirements for a successful snplex assay.
resequencing of six snp regions on wga-dna confirmed the snplex genotyping data and demonstrated the absence of amplification bias, as previously reported  <cit> .
resequencing of the snp <dig> and snp <dig> regions, belonging to the small group of snps with a distorted segregation, showed an unexpected additional polymorphism within  <dig> bp adjacent to the target esnp in the syrah genotype.
preferential ligation of one allele during probe annealing could explain the occurrence of an artificial homozygous cluster.
a large number of snp genotyping technologies have been developed in the last few years.
different aspects, such as accuracy, reproducibility and level of throughput, should be taken into account when defining the most suitable snp assay for breeding purposes.
moreover, flexibility, time and cost-effectiveness should be also considered, and in this regard, the turnaround time of the snplex analysis using a 3730xl dna analyzer  was about  <dig> min per sample.
thus  <dig>  genotypes can be theoretically generated per day .
CONCLUSIONS
unlike other snp genotyping methods used to investigate either thousands of snps in few genotypes , or few snps in thousands of genotypes , the snplex genotyping system represents a good compromise to investigate several hundred snps in a hundred or more samples at the same time.
therefore, the use of the snplex assay, coupled with a wga-dna, is a good solution for medium- to large-scale genotyping studies in well-equipped laboratories.

 in the last few years, single nucleotide polymorphisms have become the most popular genetic marker system in both animals and plants. their extraordinary abundance discovered in several genome sequencing projects, combined with recent technological improvements, makes snp markers attractive for high-throughput use in marker-assisted breeding, est mapping and the integration of genetic and physical maps. at present several snp identification methods are available such as resequencing of pcr amplicons with or without pre-screening, electronic snp discovery in expressed sequence tag and shotgun genomic libraries. in these latter cases, sequences may be computationally screened for polymorphisms to distinguish true polymorphisms from sequencing errors if sufficient redundancy is present. unlike the first generation molecular markers, such as rflps and rapds, snps can be detected through non-gel-based high-throughput assays, saving both time and money. several snp assay technologies have been developed based on various methods of allelic discrimination and detection platforms. allele-specific hybridization, primer extension, oligonucleotide ligation and invasive cleavage represent four principal allelic discrimination reactions that can be coupled with several detection methods such as fluorescence, luminescence and mass measurements. recently, significant efforts towards large-scale snp characterisation have been attempted in animals and plants with beadarray technology and the snplex™ genotyping system. the selection of an appropriate genotyping method depends on many factors including cost, potential for multiplexing and throughput, equipment, and difficulty of assay development. until recently, only a few low- and mid-throughput methods have been used for snp discovery and genotyping in grapevine. the sequencing of the highly heterozygous genome of pinot noir, clone entav made it possible to identify millions of esnps as a potential source for high-throughput genotyping methods. herein we report a successful application of the snplex genotyping system, which provided new snp-based markers anchoring the grapevine genome for future applied research programs.  snplex and data analysis on genomic dna of candidate esnps selected from non-repetitive genome contigs, passed the design rules of the snplex assay-design pipeline and were tested in f progeny of syrah × pinot noir cross and in the two parental genotypes. esnps passed the quality value using the rule-based method, with a mean of failed snps and a 98% call rate per snpset, while the remaining were discarded from further analyses. of the esnps, were false positives. of the remaining esnps, followed the 1: or 1:2: mendelian segregation ratio based on the chi-square test, whereas showed an unexpected segregation ratio. within the latter class, there were cases where one parent was heterozygous and three clusters were observed and cases where both parents were heterozygous and four clusters were detected. snplex and data analysis on whole genome amplification dna a total of esnps combined in three 48-plex snpsets were also tested on wga-dna of the same individuals. in this set, esnps that did not pass the quality value on gdna analysis were recovered on the wga-dna test, whereas three esnps that passed the quality value on gdna failed on wga-dna. thirteen esnps failed both gdna and wga-dna tests. the genotyping data were thoroughly consistent between the two analyses. resequencing analysis to validate the data obtained with the snplex assay, six regions containing snp snp snp snp snp and snp were resequenced in syrah, pinot noir and six progeny. four of them, snp snp snp and snp showed a mendelian segregation and resequencing the corresponding regions confirmed the data obtained with the snplex analysis. for snp and snp which presented an additional homozygous cluster, an unexpected snp was found within bp from the target snp in syrah and in the progeny belonging to the additional homozygous cluster. discussion in this work, we report the first snplex genotyping system application in higher plants, which allowed the development of markers anchoring the grapevine genome. to date, a few low- and mid-throughput methods based on sscp and minisequencing assay have been used for snp genotyping in grape. snp markers have also been developed based on esnp discovery in a × shotgun sequencing coverage of grapevine genome. the efficiency rate of 69% with an average call rate of 98% exceeded the level recently achieved by resequencing selected ests and bac-end sequences in previous study. although the efficiency was greatly enhanced, we observed that there were 10% systematic failed assays already detected in other snplex genotyping study and 21% false polymorphisms. this unexpectedly high rate of false polymorphisms might be due to the snplex probe design based on a preliminary stage of contig assembly where the coverage of each haplotype in some regions was not sufficient. in the wga-dna pilot study the genotyping data obtained from the gdna and wga-dna analyses were thoroughly consistent, confirming previous snp genotyping studies based on beadarray and affimetrix technologies. moreover, the number of systematic failed assays was greatly reduced, from 10% to 2%, enhancing the average efficiency rate from 69% to 80%. these results were expected since mda methods provide a large amount of pure dna with a uniform concentration among samples, meeting two basic requirements for a successful snplex assay. resequencing of six snp regions on wga-dna confirmed the snplex genotyping data and demonstrated the absence of amplification bias, as previously reported. resequencing of the snp and snp regions, belonging to the small group of snps with a distorted segregation, showed an unexpected additional polymorphism within bp adjacent to the target esnp in the syrah genotype. preferential ligation of one allele during probe annealing could explain the occurrence of an artificial homozygous cluster. a large number of snp genotyping technologies have been developed in the last few years. different aspects, such as accuracy, reproducibility and level of throughput, should be taken into account when defining the most suitable snp assay for breeding purposes. moreover, flexibility, time and cost-effectiveness should be also considered, and in this regard, the turnaround time of the snplex analysis using a 3730xl dna analyzer was about min per sample. thus genotypes can be theoretically generated per day.  unlike other snp genotyping methods used to investigate either thousands of snps in few genotypes, or few snps in thousands of genotypes, the snplex genotyping system represents a good compromise to investigate several hundred snps in a hundred or more samples at the same time. therefore, the use of the snplex assay, coupled with a wga-dna, is a good solution for medium- to large-scale genotyping studies in well-equipped laboratories. 
unlike other snp genotyping methods used to investigate thousands of snps in a few genotypes, or a few snps in around a thousand genotypes, the snplex genotyping system represents a good compromise to investigate several hundred snps in a hundred or more samples simultaneously. this approach combines robust snp detection with automated assay readout and data analysis. therefore, the use of the snplex assay, coupled with whole genome amplification, is a good solution for future applications in well-equipped laboratories. however, following completion of the sequence of the highly heterozygous genome of pinot noir, it has been possible to identify millions of electronic snps thus providing a valuable source for high-throughput genotyping methods. candidate esnps were developed from non-repetitive contigs of the assembled genome of pinot noir and tested in progeny of syrah × pinot noir cross. until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism discovery and genotyping in grapevine. the efficiency rate of 69% was enhanced to 80% when multiple displacement amplification methods were used for preparation of genomic dna for the snplex assay.
 until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism discovery and genotyping in grapevine. however, following completion of the sequence of the highly heterozygous genome of pinot noir, it has been possible to identify millions of electronic snps thus providing a valuable source for high-throughput genotyping methods.  herein we report the first application of the snplex™ genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. this approach combines robust snp detection with automated assay readout and data analysis. candidate esnps were developed from non-repetitive contigs of the assembled genome of pinot noir and tested in progeny of syrah × pinot noir cross. new snp-based markers were obtained and mapped. the efficiency rate of 69% was enhanced to 80% when multiple displacement amplification methods were used for preparation of genomic dna for the snplex assay.  unlike other snp genotyping methods used to investigate thousands of snps in a few genotypes, or a few snps in around a thousand genotypes, the snplex genotyping system represents a good compromise to investigate several hundred snps in a hundred or more samples simultaneously. therefore, the use of the snplex assay, coupled with whole genome amplification, is a good solution for future applications in well-equipped laboratories. 