Physical mapping in highly heterozygous genomes: a physical contig map of the Pinot Noir grapevine cultivar
- Simone Scalabrin1, 2,
- Michela Troggio3,
- Marco Moroldo3, 4,
- Massimo Pindo3,
- Nicoletta Felice2, 4,
- Giuseppina Coppola3,
- Giacomo Prete4,
- Giulia Malacarne3,
- Raffaella Marconi4,
- Giorgia Faes3, 4,
- Irena Jurman2, 4,
- Stella Grando3,
- Taco Jesse5,
- Cinzia Segala3, 6,
- Giorgio Valle6,
- Alberto Policriti1, 2,
- Paolo Fontana3,
- Michele Morgante2, 4Email author and
- Riccardo Velasco3
© Scalabrin et al; licensee BioMed Central Ltd. 2010
Received: 15 September 2008
Accepted: 26 March 2010
Published: 26 March 2010
Most of the grapevine (Vitis vinifera L.) cultivars grown today are those selected centuries ago, even though grapevine is one of the most important fruit crops in the world. Grapevine has therefore not benefited from the advances in modern plant breeding nor more recently from those in molecular genetics and genomics: genes controlling important agronomic traits are practically unknown. A physical map is essential to positionally clone such genes and instrumental in a genome sequencing project.
We report on the first whole genome physical map of grapevine built using high information content fingerprinting of 49,104 BAC clones from the cultivar Pinot Noir. Pinot Noir, as most grape varieties, is highly heterozygous at the sequence level. This resulted in the two allelic haplotypes sometimes assembling into separate contigs that had to be accommodated in the map framework or in local expansions of contig maps. We performed computer simulations to assess the effects of increasing levels of sequence heterozygosity on BAC fingerprint assembly and showed that the experimental assembly results are in full agreement with the theoretical expectations, given the heterozygosity levels reported for grape. The map is anchored to a dense linkage map consisting of 994 markers. 436 contigs are anchored to the genetic map, covering 342 of the 475 Mb that make up the grape haploid genome.
We have developed a resource that makes it possible to access the grapevine genome, opening the way to a new era both in grape genetics and breeding and in wine making. The effects of heterozygosity on the assembly have been analyzed and characterized by using several complementary approaches which could be easily transferred to the study of other genomes which present the same features.
With a production of 67.1 millions tons obtained from 7.9 millions hectares in 2004, grapevine (Vitis vinifera L.) is by far the most important fruit tree crop in the world http://www.oiv.org. In comparison to other fruit crops, it shows a particularly wide range of uses: fresh, dried, transformed in juice and obviously wine. At present, its importance goes beyond the economic level, being wine associated in many cases with national cultures and life style. Moreover, red wines are thought to provide health benefits thanks to secondary metabolites such as resveratrol, a strong antioxidant supposed to provide health benefits .
Despite the obvious economic relevance, most of the grape cultivars grown today were selected several centuries ago from spontaneous crosses and then vegetatively propagated. Still today, the release of new varieties is a considerably slow process. This is mainly due to some peculiar genetic and physiological features which indeed are shared with most of the tree crops and which significantly hamper the adoption of modern breeding strategies. The main difficulty for genetic analysis is due to the fact that the genome of grape is highly heterozygous, even if the species is actually mainly autogamous [2, 3]. Furthermore, grape is a perennial plant with a relatively long life cycle, which makes breeding expensive, due to space constraints, and time consuming .
Because of the lack of breeding activity, the improvements in grape production have relied mainly on agronomy, including a strong use of chemicals that may have a negative impact on human health and environmental quality. One of the main goals in grapevine breeding is therefore to obtain varieties naturally resistant to various pathogens .
As a tool for modern breeding, genome-wide physical maps are assuming a more and more central role because they are useful for many purposes. First of all, even if the technologies for genome sequencing have undergone a remarkable progress during the last years, genome assembly largely depends on physical mapping. The clone-by-clone approach always requires physical mapping, because a minimal tiling path of clones must be selected to separately sequence them . Nevertheless, also the whole-genome shotgun (WGS) strategy greatly benefits from physical data and indeed it is still unclear if it could alone be sufficient to produce a linearly ordered set of sequences . A second application of physical maps is the large-scale isolation of genes using the positional cloning approach. In fact, this is the only strategy which can be used to identify genes which can be genetically mapped, but whose function is unknown - which is true for most traits of agronomic importance . Eventually, physical maps can also be used for several other kinds of studies e.g., to compare genomes and to understand their size and complexity [5, 7, 8].
Taking into account all these aspects and considering that grape is an 'orphan species' from a genetic perspective, it is clear that an integrated physical map would be of great utility both for research stricto sensu and for practical applications, such marker-assisted breeding.
Grapevine is also attractive for future sequencing efforts, being a diploid organism which can be easily crossed and selfed, and having a relatively small genome of only about 475 Mb . Furthermore, grape has several unique features including a novel shoot architecture and non-climateric fruits producing a number of secondary metabolites such as colour pigments, tannins and flavour compounds. Finally, as a basal family of the Eurosids, the Vitaceae are also interesting for comparative studies . Thus, grape can be regarded as a model organism.
Whole-genome physical maps have been constructed for several organisms such as Caenorhabditis elegans, Drosophila melanogaster, human , mouse , and chicken . In plants, physical maps are available for Arabidopsis thaliana[16, 17], sorghum , rice , soybean , black cottonwood , apple , Prunus, and the grapevine cultivar Cabernet Sauvignon . Most of these physical maps have been obtained by adhering to different approaches, mainly by marker hybridization  and BAC fingerprinting .
Sequence Tagged Site content-based methods are laborious and require an extremely high density of markers. This piece of information is rather difficult to obtain for the large of majority of plant genomes, and especially for orphan species . On the contrary, fingerprinting-based strategies are better suited to genomes which are not well explored e.g., grape. Since these techniques are also more amenable to high-throughput processing, they have been chosen in the last years for the majority of physical mapping projects.
Due to their relevance, the techniques of fingerprinting have undergone a fast evolution during the last two decades. Indeed, a range of methods has been proposed which vary in several parameters, but especially in the detection of the fingerprinting fragments. These techniques can be divided into three groups, which are the agarose-gel based, the acrylamide-gel based and the capillary sequencer based methods. The last set of approaches, also known as High Information Content Fingerprinting (HICF), shows a substantial increase both in the throughput and in the sensitivity of the process [27, 28].
Recently, it has been debated on which HICF methodology would work best. Xu et al. evaluated five different fingerprinting techniques and concluded that a two-enzymes approach was more effective than the others. This result is rather counterintuitive, since three-, four- or five-enzymes based approaches provide more information and should be able to better discriminate between false positive and false negative errors. Indeed, according to Nelson and coworkers , the most effective protocol is the one developed by Luo et al., which indeed is based on the use of five restriction enzymes. This method has been used in this work after adapting it to grape.
Recently, important progress has been made in genomics research of various fruit trees and woody plants. The draft genome sequence of the black cottonwood tree, Populus trichocarpa, has been completed using a whole genome shotgun approach , and a physical map is available as well . Genome-wide physical maps of the apple and peach genomes have been recently released [22, 23]. To end up with, the whole genome sequences of two different genotypes of grapevine are now available, one of which based on the inbred line PN40024 , and the other one based on the heterozygous genotype Pinot Noir . Both of these sequencing efforts have been carried out following a WGS approach, therefore without the need of producing an earlier physical map.
Assessing the effects of heterozygosity on physical maps is important, because there is reason to believe that it could affect their correct assembly, similarly to what has been observed for the DNA sequence assembly in Ciona savignyi. Therefore we propose our work as a useful model for any future physical mapping effort on heterozygous genomes, which are very common among plants.
Results and discussion
Sources of BACs fingerprinted to develop the Pinot Noir physical map
Mean insert size
No. of clones fingerprinted
Total (Both libraries)
Status of the grape cv. Pinot Noir physical map assembly.
Number of clones fingerprinted
Number of clones used for map assembly
Number of singletons
Number of contigs
Unique bands in the contigs
Total physical length of the contigs (Mb)*
Number of Q clones
Number of Q contigs
In this work, the impact of DNA sequence heterozygosity on the physical map assembly was evaluated by using different approaches. First, 35 SNP markers were selected and BAC clones containing the alternative alleles were identified to ascribe them to the two haplotypes. All of the SNP markers had been genetically mapped to a unique position within the genome, to avoid the potentially confounding effect of genome duplications. However, it is to be considered that genome duplications in grapevine should not pose a big problem for the assembly of a physical map. In fact, even if grapevine appears to be an ancient hexaploid , the last round of duplication within its genome took place before the separation from black cottonwood, that is about 60-65 Myrs ago [31, 32]. Therefore, the impact of duplicated genomic regions (or homeology) on the map is likely to be very reduced in grapevine, and not as important as it is for instance in soybean .
The analysis carried out on this set of SNPs showed that both haplotypes mapped to the same contig in 13 (37%) cases ('A' class), while each haplotype mapped to a different contig in 10 (29%) cases ('B' class). In the remaining 12 (34%) cases ('C' class), BACs ascribed to the same haplotype were found on more than one contig and showed several different patterns. In 6 out of those 12 cases, the two haplotypes were found on the same contig, but one or both of the two alleles did also map to other contigs or singletons. In the other 6 cases, the two haplotypes were always found on separated contigs, but one or both of the two alleles were found on more than one contig or singleton. Altogether, these 35 polymorphic markers were found on 59 physical contigs, which corresponded to 1.7 contigs per marker. Moreover, if the same calculation was performed on the 'C' class', the value rose to 2.3 contigs per markers. The finding that allelic BAC clones i.e., deriving from each of the two haplotypes, mapped frequently to different contigs is consistent with the increased size of the physical map when compared to the estimated genome size and shows that DNA sequence heterozygosity results in significant differences in BAC fingerprints. The FPC software resolves such difficulties in a conservative mode increasing contig number. Furthermore, this evidence is perfectly in agreement with the results obtained in the physical map of black cottonwood . Even when the BAC clones from the two haplotypes assembled in a single contig (Figure 1a, b) their relative positioning within the contig was often not accurate, with the clones from each haplotype locally assembling separately rather than overlapping each other. This local assembly problem, which we named "scissoring effect", was not observed in the black cottonwood assembly.
Computer simulations of the effects of haplotype polymorphism on contig assembly using FPC.
% different fingerprint bands
% average sequence divergence
Consensus band units for entire region
Map coverage of the region
Number of contigs
% Questionable clones
1,172.5 ± 39.9
1.0 ± 0.0
1.1 ± 0.3
0.0 ± 0.0
1,252.4 ± 89.0
1.1 ± 0.1
1.2 ± 0.4
6.0 ± 4.9
1,681.6 ± 129.4
1.4 ± 0.1
2.2 ± 1.0
18.8 ± 8.5
1,717.7 ± 206.5
1.4 ± 0.2
2.2 ± 0.9
24.7 ± 9.2
Results of BAC pools screening with molecular markers.
Markers not assigned to BAC clones*
False positive or not fingerprinted BAC clones
Not amplified in one of the six pools or over-represented in BAC clones
Markers assigned to BAC clones:
Class I: identified two or more BACs assembled in a single contig
Class II: identified only one BAC in a single contig
Class III: identified singletons (unassembled BACs)
Class IV: identified multiple BACs assembled into distinct contigs
Statistics of the grape cv. Pinot Noir physical map by linkage group
No. of contigs
Average contig size (Kb)
Total contigs assigned to markers
Chimeric clones detected by BES alignment versus the genomic sequence (linkage groups between parentheses) and by counting putative intra-plate contaminations (high number of clones from a single plate, in parentheses, in the same contig)
Putative chimeric clone(s)
BES alignment (7, 14)
BES alignment (2, 7)
BES alignment (4, 5)
BES alignment (2, 18)
BES alignment (1, 14)
BES alignment (2,18)
BES alignment (6, 16)
BES alignment (4, 14)
BES alignment (4, 16)
All clones from plate 1044
BES alignment (18, 19), Intra-plate (1039)
BES alignment (7, 10, 14)
We have developed a resource that facilitates the access to the grapevine genome in several ways. By one hand, this tool can be used to easily create a link between genetic maps and genomic sequences, thus facilitating tasks such as positional cloning or QTL mapping. By another point of view it could also become useful for comparative purposes when studying other species belonging to the family of Vitaceae.
The second relevant aspect which has been studied in this work is the effect of heterozygosity on the construction of fingerprinting-based physical maps. Experimental and in silico approaches have been undertaken and have given comparable results, thus representing an example of how the use of simulated data can help the study of genomes. Furthermore, the description of the effects of heterozygosity in physical mapping could be particularly useful for further studies of genomes showing the same features.
It looks like the assembly could be improved by the utilization of new map assembly algorithms that explicitly deal with the presence of two haplotypes regardless of the levels and patterns of heterozygosity.
BAC library construction and BAC pooling
Hind III partially digested HMW DNA from V. vinifera cv. Pinot Noir clone 115 was used to construct two libraries. The first was obtained as described by Adam Blondon et al. and consisted of 23,424 clones (4.6× genome coverage) stored into 384-well microplates. The average insert size and the percentages of empty clones and plastidial DNA were controlled as well . The second library was produced at Keygene (Wageningen, The Netherlands). DNA was ligated into the pIndigoBAC vector and ligation mixes with less than 4% of empty clones, less than 4% of cpDNA contamination and insert size >125 kb were selected. After transformation into E. coli strain DH10β, 26.112 colonies were picked up (6.8× genome coverage). In total, 49,104 clones were available for fingerprinting. Average insert sizes for the two libraries are described in Table 1.
BAC pooling followed the strategy proposed by Klein et al. 24,576 BAC clones (5× genome coverage) were arranged in a stack which was sampled in six distinct ways and a total of 184 pools were obtained.
BAC fingerprinting and BAC-end sequencing
BAC DNA was isolated and fingerprinted adapting to grape the technology already developed for wheat and rice . BAC clones maintained in 384-well microplates were preinoculated in 384-well microplates containing 70 μl 2× LB medium plus chloramphenicol (12.5 mg/ml) per well, inoculated in 96-well plates containing 1.2 ml of the same medium per well, and grown at 37°C with shaking at 250 rpm for 10-15 h. BAC DNA was isolated using the 96-well Unifilter plates (Whatman 7700-0062) following the procedure described by Luo and coworkers  and dissolved in 20 μl of ddH2O.
A total of 5 μl of a solution containing 5 units each of Bam HI, Eco RI, Nde I, Xba I, and Hae III restriction enzymes; 1× NEBuffer 2 (New England Biolabs, Boston, MA); 2 μg BSA; 2 μg DNase-free RNase; and 0.1% β-mercaptoethanol was added to 15 μL of the BAC DNA solution and the digestion was carried out at 37°C for 3 h in a 384-well PCR plate. Subsequently, 3 μL of labelling solution made up of 1.5 μL SNapShot Multiplex Ready Reaction Mix (Applied Biosystems, Foster City, CA), 0.8 μL Tris-HCl 100 mM, pH = 9.0, and 0.7 μL ddH2O were added to each well and the plates were incubated at 65°C for 1 h.
The BAC fingerprinting reaction was precipitated in ethanol, resuspended in 10 μL of ddH2O and filtered through Sephadex using the 384-well genCLEAN plates (Genetix, New Milton, UK). After cleaning, DNA was resuspended again in 10 μL of ddH2O and a mixture of 10.98 μL of Hi-Di formamide and 0.02 μL of Genescan LIZ-500 internal size standard (Applied Biosystems, Foster City, CA) was added to each well. The samples were then denaturated and loaded into an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA).
End sequencing of 19,957 BAC clones was performed using 2.5 μL of BAC DNA and 1.0 of Big Dye 3.1 Terminator Ready Reaction Mix (Applied Biosystems, Foster City, CA), for a total reaction volume of 10 μL. Traces were analysed by a pipeline of programs, including Phred, RepeatMasker, Mummer, Cap3, and CrossMatch for quality clipping, clustering, and repeats identification (unpublished data). Some of the unique sequences were then used for BAC anchoring to the linkage groups of the genetic map.
Data processing and assembly of the physical map
The ABI GeneMapper 3.5 software (Applied Biosystems, Foster City, CA) was first used to analyze all electrochromatograms and to produce a text file (containing areas, heights, and sizes for each peak) for each BAC fingerprint. Then, 809 ribosomal and 941 chloroplastic contaminated clones were removed. Ribosomal and chloroplastic clones were detected through a comparison to previously built-in ribosomal and chloroplastic fingerprint patterns and by blasting the 30,158 BAC-ends versus the ribosomal (e-value 1E-30) and chloroplastic (e-value 1E-200) sequences inferred from the genome assemblies. Then, remaining clones were processed by FPB , a PERL script to distinguish between peaks originating from BAC clone restriction fragments and background peaks (e.g., E. coli genomic DNA contamination or machine noise) using an iterative procedure, to remove empty clones and vector bands and to convert data to FPC format as in Genoprofiler. Finally, 384 and 96 cross-well contaminations were detected with the use of the software GenoProfiler : 1471 adjacent clones sharing at least 50% of their bands were considered cross-well contaminated and removed before the assembly. A further analysis to prove that the in silico procedure was sufficient to remove contaminations was performed on contig 71 that is apparently highly contaminated as 19 of its 38 clones originated from only 9 plates. Two analysis were carried out. Initially, BAC-end sequencing and corresponding alignment on the PN40024 sequence revealed that in all cases but 2 there was no contamination since sequences were aligning in a single region of chromosome 1; only two reads had the best hit on a different location (with their mate on the correct place) [see Additional file 1]. Successively, four clones with no sequence or with uncertain BES alignment were picked up from glycerol stock and inoculated in 2× LB Medium with chloramphenicol (12.5 mg/ul) over night, after that each clone was streaked onto 2× LB medium agarose plate with chloramphenicol and grown over night. Six single colonies for each clone were picked and inoculated in 2× LB medium with chloramphenicol over night individually. BAC DNA of all clones were isolated and tested by capillary electrophoresis fingerprinting. Fingerprints produced for each of the six single colonies within each of the individual BAC clone resulted identical.
The contig assembly was carried out based on the software FPC 8.9 and following the approach suggested by Nelson et al.. Tolerance was set at 0.4 bp and for the initial build the cutoff value was set at 1E-50. The map was then further assembled in an iterative way, through 6 rounds at successively less stringent cutoffs. Each round consisted in: 1) a 3-step DQing process starting at the same cutoff of the previous assembly and then decreasing the cutoff by 105 at every successive step and 2) an automatic end-joining step performed increasing the cutoff value of the previous assembly by 105 times. Finally, contigs were tested with internally developed scripts, to detect intra-plate contaminations: only one contig was seriously affected by such a problem (see Table 6, contig 2207).
In silico assessment of the effects of heterozygosity on BAC contig assembly
We started from the Consensus Band (CB) map of a high quality assembled contig containing 1192 bands. We assumed this to represent the ordered fingerprint bands for the region from a single chromosome/haplotype. We produced the expected CB map of the same region from the homologous/allelic chromosome/haplotype by randomly modifying a certain percentage of bands corresponding to a certain level of sequence divergence. As a first approximation each band produced in our fingerprinting method can be considered to be defined by 10 nucleotides and therefore a randomly distributed sequence divergence of 1% between the two haplotypes is expected to produce a difference in 10% of the fingerprint bands obtained from the two chromosomes/haplotypes. For each of the two chromosomes BACs were created randomly to provide 5× coverage by defining an average insert size (120 bands) and a range of variation (from 50 to 190 bands). Ten replicates of the BACs from the two haplotypes were created for each sequence divergence condition. The assembly was carried out iteratively, as already described for the map, starting from a cutoff value of 1E-50. Averages of the different estimated parameters were then obtained from the 10 replicates.
Integration between genetic and physical maps
A linkage map of the V. vinifera L. cross Syrah x Pinot noir  was the basis to anchor genetically fingerprinted BAC clones. In the genetic map, 994 markers were positioned with the help of 94 F1 hybrid individuals and grouped in 19 linkage groups covering a total length of 1,245 cM. Linkage group assignment and ordering of loci was established based on TMAP software  that finds the maximum likelihood map using an error-compensating model. Details of linkage map construction are provided elsewhere .
The integration between physical and genetic maps was carried out in two steps. First, 133 simple sequence repeats (SSRs) and 174 expressed sequence tags (ESTs) mapped markers were used to screen the BAC pools following a hot start amplification strategy . After adding SYBR Gold, samples were run on 1.5% agarose gel. BAC clones hosting SSR and EST markers were identified by a Unix-based application with a web interface. Additional anchor points were provided by screening the BAC pools with the 15 unique amplified fragment length polymorphism combinations  used to identify 162 mapped AFLPs. Preamplification and selective amplification were performed as for linkage analysis. AFLP amplification products from BAC pools were analyzed on acrylamide gels along with amplification products from the two parents and the mapping population as a control (AFLP Quant-Pro, Keygene, Wageningen, NL). BACs containing AFLPs were identified as for the other markers. Finally, the genetic position of 316 SNP markers developed from the BAC-ends provided additional anchoring information.
Amplified Fragment Length Polymorphism
Bacterial Artificial Chromosome
Expressed Sequence Tag
High Information Content Fingerprinting
Quantitative Trait Locus
Single Nucleotide Polymorphism
Simple Sequence Repeat
Whole Genome Shotgun.
This work was supported by the "Grapevine Physical Mapping" project funded by the Provincia Autonoma di Trento. The work on heterozygosity simulations was supported by a MIUR-FIRB grant to MM and by FIRB grant RBNE03B8KK to SS.
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