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Physical mapping in highly heterozygous genomes: a physical contig map of the Pinot Noir grapevine cultivar.

Scalabrin S, Troggio M, Moroldo M, Pindo M, Felice N, Coppola G, Prete G, Malacarne G, Marconi R, Faes G, Jurman I, Grando S, Jesse T, Segala C, Valle G, Policriti A, Fontana P, Morgante M, Velasco R - BMC Genomics (2010)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Genomica Applicata, Parco Scientifico e Tecnologico di Udine Luigi Danieli, Via J Linussio 51, 33100 Udine, Italy.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Effect of heterozygosity on the assembly of the physical map. a/ and b/ CB Maps of two real contigs of the grape physical map ('a' corresponds to contig number 69 and 'b' to contig number 354) showing the so called 'scissoring effect', one of the main consequences of heterozygosity. On both contigs a polymorphic SNP marker has been physically mapped by BAC-pooling first, followed by PCR determination of the BAC clones containing each of the two alleles. The clones on which one allele maps have been highlighted in red and the ones on which the other allele maps in blue. It is interesting to observe that the two alleles of the same SNP marker map onto different clones which are not overlapping each other. The problem which affects the assembly of these two contigs has been called 'scissoring effect'. Basically, the clones within a contig tend to split apart, with the ones belonging to one allele at one extremity and the ones belonging to the other allele at the other extremity. As a consequence, the affected contigs show an expansion in the CB map size. c/ and d/ Two plots depicting simulated contigs from the in silico simulation at 42% sequence divergence, where the clones belonging to one allele have been highlighted in orange and the ones belonging to the other allele in blue. In 'c' the 'scissoring effect' is particularly evident on the right extremity, while the other part of the contig shows a more complex situation. Indeed, the scissoring effect can be locally more or less important and this is thought to be related to the variation in the levels of heterozygosity along the contig/chromosome. This situation is particularly consistent with the pictures 'a' and 'b'. The contig displayed in 'd' shows instead a different situation, being made up by clones which belong only to one allele (in orange). This scenario is consistent with the cases of allele testing in which the two alleles hybridize onto separate contigs (see text).
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Figure 1: Effect of heterozygosity on the assembly of the physical map. a/ and b/ CB Maps of two real contigs of the grape physical map ('a' corresponds to contig number 69 and 'b' to contig number 354) showing the so called 'scissoring effect', one of the main consequences of heterozygosity. On both contigs a polymorphic SNP marker has been physically mapped by BAC-pooling first, followed by PCR determination of the BAC clones containing each of the two alleles. The clones on which one allele maps have been highlighted in red and the ones on which the other allele maps in blue. It is interesting to observe that the two alleles of the same SNP marker map onto different clones which are not overlapping each other. The problem which affects the assembly of these two contigs has been called 'scissoring effect'. Basically, the clones within a contig tend to split apart, with the ones belonging to one allele at one extremity and the ones belonging to the other allele at the other extremity. As a consequence, the affected contigs show an expansion in the CB map size. c/ and d/ Two plots depicting simulated contigs from the in silico simulation at 42% sequence divergence, where the clones belonging to one allele have been highlighted in orange and the ones belonging to the other allele in blue. In 'c' the 'scissoring effect' is particularly evident on the right extremity, while the other part of the contig shows a more complex situation. Indeed, the scissoring effect can be locally more or less important and this is thought to be related to the variation in the levels of heterozygosity along the contig/chromosome. This situation is particularly consistent with the pictures 'a' and 'b'. The contig displayed in 'd' shows instead a different situation, being made up by clones which belong only to one allele (in orange). This scenario is consistent with the cases of allele testing in which the two alleles hybridize onto separate contigs (see text).

Mentions: Here we report a BAC-based physical map of the grape genome and its integration with the genetic map, a molecular tool which will change the approach to the genetics and breeding of this crop. As already stated, grapevine cultivars are highly heterozygous [3,24]. Physical maps constructed till now present almost no (e.g., mouse and Arabidopsis) or very low heterozygosity (e.g., human). The only exceptions are black cottonwood, Prunus, apple, and the Cabernet Sauvignon grapevine cultivar. Black cottonwood and apple maps are based on agarose fingerprints and the effects of heterozygosity with such method are smoothened by the higher tolerance allowed in the assembly. Beside that, a specific study about the effects of heterozygosity on the map assembly has been performed only in the case of black cottonwood [21]. On the other hand, the map of Prunus has been obtained with the same fingerprinting method chosen for grape, but other differences make the comparison quite difficult. In fact, the mapping effort in peach has not been completed yet and the clones fingerprinted so far (4.3× genome equivalents), are biased towards the expressed regions of the genome. Moreover, two BAC libraries were used in this case, one obtained from a diploid genotype and the other from a haploid one, which could have reduced the impact of heterozygosity. Finally, concerning heterozygosity, the map of Cabernet Sauvignon, obtained with the same protocol as the Pinot Noir map, lead to similar results: incorrect order of the BAC clones within a contig, producing apparent duplications of loci in the physical map and assembly of BAC clones corresponding to two different haplotypes into separate contigs. See Figure 1 and the discussion below for further details, including an in silico simulation related to the effects of heterozygosity on a map assembly.


Physical mapping in highly heterozygous genomes: a physical contig map of the Pinot Noir grapevine cultivar.

Scalabrin S, Troggio M, Moroldo M, Pindo M, Felice N, Coppola G, Prete G, Malacarne G, Marconi R, Faes G, Jurman I, Grando S, Jesse T, Segala C, Valle G, Policriti A, Fontana P, Morgante M, Velasco R - BMC Genomics (2010)

Effect of heterozygosity on the assembly of the physical map. a/ and b/ CB Maps of two real contigs of the grape physical map ('a' corresponds to contig number 69 and 'b' to contig number 354) showing the so called 'scissoring effect', one of the main consequences of heterozygosity. On both contigs a polymorphic SNP marker has been physically mapped by BAC-pooling first, followed by PCR determination of the BAC clones containing each of the two alleles. The clones on which one allele maps have been highlighted in red and the ones on which the other allele maps in blue. It is interesting to observe that the two alleles of the same SNP marker map onto different clones which are not overlapping each other. The problem which affects the assembly of these two contigs has been called 'scissoring effect'. Basically, the clones within a contig tend to split apart, with the ones belonging to one allele at one extremity and the ones belonging to the other allele at the other extremity. As a consequence, the affected contigs show an expansion in the CB map size. c/ and d/ Two plots depicting simulated contigs from the in silico simulation at 42% sequence divergence, where the clones belonging to one allele have been highlighted in orange and the ones belonging to the other allele in blue. In 'c' the 'scissoring effect' is particularly evident on the right extremity, while the other part of the contig shows a more complex situation. Indeed, the scissoring effect can be locally more or less important and this is thought to be related to the variation in the levels of heterozygosity along the contig/chromosome. This situation is particularly consistent with the pictures 'a' and 'b'. The contig displayed in 'd' shows instead a different situation, being made up by clones which belong only to one allele (in orange). This scenario is consistent with the cases of allele testing in which the two alleles hybridize onto separate contigs (see text).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Effect of heterozygosity on the assembly of the physical map. a/ and b/ CB Maps of two real contigs of the grape physical map ('a' corresponds to contig number 69 and 'b' to contig number 354) showing the so called 'scissoring effect', one of the main consequences of heterozygosity. On both contigs a polymorphic SNP marker has been physically mapped by BAC-pooling first, followed by PCR determination of the BAC clones containing each of the two alleles. The clones on which one allele maps have been highlighted in red and the ones on which the other allele maps in blue. It is interesting to observe that the two alleles of the same SNP marker map onto different clones which are not overlapping each other. The problem which affects the assembly of these two contigs has been called 'scissoring effect'. Basically, the clones within a contig tend to split apart, with the ones belonging to one allele at one extremity and the ones belonging to the other allele at the other extremity. As a consequence, the affected contigs show an expansion in the CB map size. c/ and d/ Two plots depicting simulated contigs from the in silico simulation at 42% sequence divergence, where the clones belonging to one allele have been highlighted in orange and the ones belonging to the other allele in blue. In 'c' the 'scissoring effect' is particularly evident on the right extremity, while the other part of the contig shows a more complex situation. Indeed, the scissoring effect can be locally more or less important and this is thought to be related to the variation in the levels of heterozygosity along the contig/chromosome. This situation is particularly consistent with the pictures 'a' and 'b'. The contig displayed in 'd' shows instead a different situation, being made up by clones which belong only to one allele (in orange). This scenario is consistent with the cases of allele testing in which the two alleles hybridize onto separate contigs (see text).
Mentions: Here we report a BAC-based physical map of the grape genome and its integration with the genetic map, a molecular tool which will change the approach to the genetics and breeding of this crop. As already stated, grapevine cultivars are highly heterozygous [3,24]. Physical maps constructed till now present almost no (e.g., mouse and Arabidopsis) or very low heterozygosity (e.g., human). The only exceptions are black cottonwood, Prunus, apple, and the Cabernet Sauvignon grapevine cultivar. Black cottonwood and apple maps are based on agarose fingerprints and the effects of heterozygosity with such method are smoothened by the higher tolerance allowed in the assembly. Beside that, a specific study about the effects of heterozygosity on the map assembly has been performed only in the case of black cottonwood [21]. On the other hand, the map of Prunus has been obtained with the same fingerprinting method chosen for grape, but other differences make the comparison quite difficult. In fact, the mapping effort in peach has not been completed yet and the clones fingerprinted so far (4.3× genome equivalents), are biased towards the expressed regions of the genome. Moreover, two BAC libraries were used in this case, one obtained from a diploid genotype and the other from a haploid one, which could have reduced the impact of heterozygosity. Finally, concerning heterozygosity, the map of Cabernet Sauvignon, obtained with the same protocol as the Pinot Noir map, lead to similar results: incorrect order of the BAC clones within a contig, producing apparent duplications of loci in the physical map and assembly of BAC clones corresponding to two different haplotypes into separate contigs. See Figure 1 and the discussion below for further details, including an in silico simulation related to the effects of heterozygosity on a map assembly.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Genomica Applicata, Parco Scientifico e Tecnologico di Udine Luigi Danieli, Via J Linussio 51, 33100 Udine, Italy.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH