Development of the GlutEnSeq capture system for sequencing gluten gene families in hexaploid bread wheat with deletions or mutations induced by γ-irradiation or CRISPR/Cas9
Jouanin, Aurélie ; Borm, Theo ; Boyd, Lesley A. ; Cockram, James ; Leigh, Fiona ; Santos, Bruno A.C.M. ; Visser, Richard G.F. ; Smulders, Marinus J.M. - \ 2019
Journal of Cereal Science 88 (2019). - ISSN 0733-5210 - p. 157 - 166.
Breeding - Coeliac - Disease - Editing - Gene - Gliadin - Mutation
We developed an in-solution gluten exome capture system called GlutEnSeq (Gluten gene Enrichment and Sequencing), covering the sequence variation of thousands of prolamin genes from various Triticeae species and cultivars. We assessed the efficacy of this capture system in hexaploid wheat (Triticum aestivum L.) using Illumina sequencing. On-target regions were determined based on the Chinese Spring (CS) reference genome sequence. Gluten gene sequences were generally enriched around 10,000-fold. The loss of gluten genes in CS deletion line 1BS-19/6DS-4 was detected as absence of gluten gene coverage on chromosomes 1B and Un (containing the Unmapped α-gliadin genes of chromosome 6D). Two γ-irradiated lines of cultivar Paragon, shown to be affected in their gliadin protein profile, were found to contain homozygous deletions for the α-gliadins on 6A and the γ-gliadins on 1B. Four Fielder CRISPR/Cas9 gliadin gene-edited lines revealed homozygous deletions of the γ-gliadins on 1B and heterozygous deletions for the α-gliadins on 6A. We also detected a decrease of gluten gene coverage within some gluten genes. The bioinformatics pipeline developed here will be further optimised to enable characterisation of small indels within individual gluten genes, in order to fully analyse CRISPR/Cas9 mutant lines for their decrease in immunogenicity for Coeliac patients.
Comparative Subsequence Sets Analysis (CoSSA) is a robust approach to identify haplotype specific SNPs; Mapping and pedigree analysis of a potato wart disease resistance gene Sen3
Prodhomme, Charlotte ; Esselink, Danny ; Borm, Theo ; Visser, Richard G.F. ; Eck, Herman J. Van; Vossen, Jack H. - \ 2019
Plant Methods 15 (2019)1. - ISSN 1746-4811
Bulked segregant analysis (BSA) - Diagnostic markers - k-mers - Potato wart disease - Reference genome - Whole genome sequencing (WGS)
Background: Standard strategies to identify genomic regions involved in a specific trait variation are often limited by time and resource consuming genotyping methods. Other limiting pre-requisites are the phenotyping of large segregating populations or of diversity panels and the availability and quality of a closely related reference genome. To overcome these limitations, we designed efficient Comparative Subsequence Sets Analysis (CoSSA) workflows to identify haplotype specific SNPs linked to a trait of interest from Whole Genome Sequencing data. Results: As a model, we used the resistance to Synchytrium endobioticum pathotypes 2, 6 and 18 that co-segregated in a tetraploid full sib population. Genomic DNA from both parents, pedigree genotypes, unrelated potato varieties lacking the wart resistance traits and pools of resistant and susceptible siblings were sequenced. Set algebra and depth filtering of subsequences (k-mers) were used to delete unlinked and common SNPs and to enrich for SNPs from the haplotype(s) harboring the resistance gene(s). Using CoSSA, we identified a major and a minor effect locus. Upon comparison to the reference genome, it was inferred that the major resistance locus, referred to as Sen3, was located on the north arm of chromosome 11 between 1,259,552 and 1,519,485 bp. Furthermore, we could anchor the unanchored superscaffold DMB734 from the potato reference genome to a synthenous interval. CoSSA was also successful in identifying Sen3 in a reference genome independent way thanks to the de novo assembly of paired end reads matching haplotype specific k-mers. The de novo assembly provided more R haplotype specific polymorphisms than the reference genome corresponding region. CoSSA also offers possibilities for pedigree analysis. The origin of Sen3 was traced back until Ora. Finally, the diagnostic power of the haplotype specific markers was shown using a panel of 56 tetraploid varieties. Conclusions: CoSSA is an efficient, robust and versatile set of workflows for the genetic analysis of a trait of interest using WGS data. Because the WGS data are used without intermediate reads mapping, CoSSA does not require the use of a reference genome. This approach allowed the identification of Sen3 and the design of haplotype specific, diagnostic markers.
A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits
Hibrand Saint-Oyant, L. ; Ruttink, T. ; Hamama, L. ; Kirov, I. ; Lakhwani, D. ; Zhou, N.N. ; Bourke, P.M. ; Daccord, N. ; Leus, L. ; Schulz, D. ; Geest, H. van de; Hesselink, T. ; Laere, K. Van; Debray, K. ; Balzergue, S. ; Thouroude, T. ; Chastellier, A. ; Jeauffre, J. ; Voisine, L. ; Gaillard, S. ; Borm, T.J.A. ; Arens, P. ; Voorrips, R.E. ; Maliepaard, C. ; Neu, E. ; Linde, M. ; Paslier, M.C. Le; Bérard, A. ; Bounon, R. ; Clotault, J. ; Choisne, N. ; Quesneville, H. ; Kawamura, K. ; Aubourg, S. ; Sakr, S. ; Smulders, M.J.M. ; Schijlen, E. ; Bucher, E. ; Debener, T. ; Riek, J. De; Foucher, F. - \ 2018
Nature Plants 4 (2018). - ISSN 2055-026X - p. 473 - 484.
Rose is the world’s most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line (‘HapOB’) from Rosa chinensis ‘Old Blush’ and generated a rose genome assembly anchored to seven pseudo-chromosomes (512 Mb with N50 of 3.4 Mb and 564 contigs). The length of 512 Mb represents 90.1–96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.
De Novo Assembly of Complete Chloroplast Genomes from Non-model Species Based on a K-mer Frequency-Based Selection of Chloroplast Reads from total DNA Sequences.
Izan, Shairul ; Esselink, G. ; Visser, R.G.F. ; Smulders, M.J.M. ; Borm, T.J.A. - \ 2017
Frontiers in Plant Science 8 (2017). - ISSN 1664-462X - 13 p.
Whole Genome Shotgun (WGS) sequences of plant species often contain an abundance of reads that are derived from the chloroplast genome. Up to now these reads have generally been identified and assembled into chloroplast genomes based on homology to chloroplasts from related species. This re-sequencing approach may select against structural differences between the genomes especially in non-model species for which no close relatives have been sequenced before. The alternative approach is to de novo assemble the chloroplast genome from total genomic DNA sequences. In this study, we used k-mer frequency tables to identify and extract the chloroplast reads from the WGS reads and assemble these using a highly integrated and automated custom pipeline. Our strategy includes steps aimed at optimizing assemblies and filling gaps which are left due to coverage variation in the WGS dataset. We have successfully de novo assembled three complete chloroplast genomes from plant species with a range of nuclear genome sizes to demonstrate the universality of our approach: Solanum lycopersicum (0.9 Gb), Aegilops tauschii (4 Gb) and Paphiopedilum henryanum (25 Gb). We also highlight the need to optimize the choice of k and the amount of data used. This new and cost-effective method for de novo short read assembly will facilitate the study of complete chloroplast genomes with more accurate analyses and inferences, especially in non-model plant genomes.
|The development of a high-throughput marker platform for genome-wide mapping of NB-LRR resistance genes in potato.
Holterman, M.H.M. ; Bakker, E.H. ; Elsen, S.J.J. van den; Borm, T.J.A. ; Pomp, H. ; Sterken, M.G. ; Helder, J. ; Goverse, A. - \ 2017
Potato is one of the world’s most important food crops and pathogens cause large losses annually. The use of resistant potato varieties is one of the most important means of controlling the impact of these pathogens. However, not all genes responsible for these resistances are known. The majority of known pathogen resistances in potato are conferred by single dominant R genes encoding nucleotide binding leucine-rich-repeat (NB-LRR) proteins. NB-LRR genes occur in clusters throughout the genome and with the aid of the reference potato genome these clusters could be identified. Our aim was to establish a strategy based on cluster specific markers for genome-wide identification of NB-LRR genes conferring disease resistance to various pathogens in potato. For each gene cluster a primer pair was developed to specifically amplify a pool of fragments from the NB-LRR genes present in a single cluster. These primers were combined in ten-plex PCR setups. These pools of amplicons were subsequently sequenced using the Illumina MiSeq sequencing platform. A bioinformatics pipeline was set up to identify all the read variants present in the set of gene clusters and to convert them into informative markers to find correlations with known and unknown resistance traits in a panel of potato varieties or mapping populations. One of the major pathogens on potato are potato cyst nematodes (PCN’s; Globodera rostochiensis and G. pallida). Known R genes, such as the Gpa2 gene conferring resistance to G. pallida, will be used to validate the approach, showing its potential as a genome-wide HTP marker platform to find novel NB-LRR genes linked to disease resistances in potato.
The genome of Chenopodium quinoa
Jarvis, D.E. ; Shwen Ho, Yung ; Lightfoot, Damien J. ; Schmöckel, Sandra M. ; Li, Bo ; Borm, T.J.A. ; Ohyanagi, Hajime ; Mineta, Katsuhiko ; Mitchell, Craig T. ; Saber, Noha ; Kharbatia, Najeh M. ; Rupper, Ryan R. ; Sharp, Aaron R. ; Dally, Nadine ; Boughton, Berin A. ; Woo, Yong H. ; Gao, Ge ; Schijlen, E.G.W.M. ; Guo, Xiujie ; Momin, Afaque A. ; Negräo, Sónia ; Al-Babili, Salim ; Gehring, Christoph ; Roessner, Ute ; Jung, Christian ; Murphy, Kevin ; Arold, Stefan T. ; Gojobori, Takashi ; Linden, C.G. van der; Loo, E.N. van; Jellen, Eric N. ; Maughan, Peter J. ; Tester, Mark - \ 2017
Nature 542 (2017)7641. - ISSN 0028-0836 - p. 307 - 312.
Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.
A Phylogenetic Analysis of Chloroplast Genomes Elucidates the Relationships of the Six Economically Important Brassica Species Comprising the Triangle of U
Li, Peirong ; Zhang, Shujiang ; Li, Fei ; Zhang, Shifan ; Zhang, Hui ; Wang, Xiaowu ; Sun, Rifei ; Bonnema, Guusje ; Borm, Theo J.A. - \ 2017
Frontiers in Plant Science 8 (2017). - ISSN 1664-462X
The Brassica genus comprises many economically important worldwide cultivated crops. The well-established model of the Brassica genus, U’s triangle, consists of three basic diploid plant species (Brassica rapa, Brassica oleracea, and Brassica nigra) and three amphidiploid species (Brassica napus, Brassica juncea, and Brassica carinata) that arose through interspecific hybridizations. Despite being extensively studied because of its commercial relevance, several aspects of the origin of the Brassica species and the relationships within and among these six species still remain open questions. Here, we successfully de novo assembled 60 complete chloroplast genomes of Brassica genotypes of all six species. A complete map of the single nucleotide variants and insertions and deletions in the chloroplast genomes of different Brassica species was produced. The chloroplast genome consists of a Large and a Small Single Copy (LSC and SSC) region between two inverted repeats, and while these regions of chloroplast genomes have very different molecular evolutionary rates, phylogenetic analyses of different regions yielded no contradicting topologies and separated the Brassica genus into four clades. B. carinata and B. juncea share their chloroplast genome with one of their hybridization donors B. nigra and B. rapa, respectively, which fits the U model. B. rapa, surprisingly, shows evidence of two types of chloroplast genomes, with one type specific to some Italian broccoletto accessions. B. napus clearly has evidence for two independent hybridization events, as it contains either B. rapa chloroplast genomes. The divergence estimation suggests that B. nigra and B. carinata diverged from the main Brassica clade 13.7 million years ago (Mya), while B. rapa and B. oleracea diverged at 2.18 Mya. The use of the complete chloroplast DNA sequence not only provides insights into comparative genome analysis but also paves the way for a better understanding of the phylogenetic relationships within the Brassica genus.
Genome resequencing and comparative variome analysis in a Brassica rapa and Brassica oleracea collection
Cheng, Feng ; Wu, Jian ; Cai, Chengcheng ; Fu, Lixia ; Liang, Jianli ; Borm, Theo ; Zhuang, Mu ; Zhang, Yangyong ; Zhang, Fenglan ; Bonnema, Guusje ; Wang, Xiaowu - \ 2016
Scientific Data 3 (2016). - ISSN 2052-4463
The closely related species Brassica rapa and B. oleracea encompass a wide range of vegetable, fodder and oil crops. The release of their reference genomes has facilitated resequencing collections of B. rapa and B. oleracea aiming to build their variome datasets. These data can be used to investigate the evolutionary relationships between and within the different species and the domestication of the crops, hereafter named morphotypes. These data can also be used in genetic studies aiming at the identification of genes that influence agronomic traits. We selected and resequenced 199 B. rapa and 119 B. oleracea accessions representing 12 and nine morphotypes, respectively. Based on these resequencing data, we obtained 2,249,473 and 3,852,169 high quality SNPs (single-nucleotide polymorphisms), as well as 303,617 and 417,004 InDels for the B. rapa and B. oleracea populations, respectively. The variome datasets of B. rapa and B. oleracea represent valuable resources to researchers working on evolution, domestication or breeding of Brassica vegetable crops.
Subgenome parallel selection is associated with morphotype diversification and convergent crop domestication in Brassica rapa and Brassica oleracea
Cheng, Feng ; Sun, Rifei ; Hou, Xilin ; Zheng, Hongkun ; Zhang, Fenglan ; Zhang, Yangyong ; Liu, Bo ; Liang, Jianli ; Zhuang, Mu ; Liu, Yunxia ; Liu, Dongyuan ; Wang, Xiaobo ; Li, Pingxia ; Liu, Yumei ; Lin, Ke ; Bucher, Johan ; Zhang, Ningwen ; Wang, Yan ; Wang, Hui ; Deng, Jie ; Liao, Yongcui ; Wei, Keyun ; Zhang, Xueming ; Fu, Lixia ; Hu, Yunyan ; Liu, Jisheng ; Cai, Chengcheng ; Zhang, Shujiang ; Zhang, Shifan ; Li, Fei ; Zhang, Hui ; Zhang, Jifang ; Guo, Ning ; Liu, Zhiyuan ; Liu, Jin ; Sun, Chao ; Ma, Yuan ; Zhang, Haijiao ; Cui, Yang ; Freeling, Micheal R. ; Borm, Theo ; Bonnema, Guusje ; Wu, Jian ; Wang, Xiaowu - \ 2016
Nature Genetics 48 (2016)10. - ISSN 1061-4036 - p. 1218 - 1224.
Brassica species, including crops such as cabbage, turnip and oilseed, display enormous phenotypic variation. Brassica genomes have all undergone a whole-genome triplication (WGT) event with unknown effects on phenotype diversification. We resequenced 199 Brassica rapa and 119 Brassica oleracea accessions representing various morphotypes and identified signals of selection at the mesohexaploid subgenome level. For cabbage morphotypes with their typical leaf-heading trait, we identified four subgenome loci that show signs of parallel selection among subgenomes within B. rapa, as well as four such loci within B. oleracea. Fifteen subgenome loci are under selection and are shared by these two species. We also detected strong subgenome parallel selection linked to the domestication of the tuberous morphotypes, turnip (B. rapa) and kohlrabi (B. oleracea). Overall, we demonstrated that the mesohexaploidization of the two Brassica genomes contributed to their diversification into heading and tuber-forming morphotypes through convergent subgenome parallel selection of paralogous genes.
Homologues of potato chromosome 5 show variable collinearity in the euchromatin, but dramatic absence of synteny in the pericentromeric heterochromatin
Boer, J.M. de; Datema, E. ; Tang, X. ; Borm, T.J.A. ; Bakker, E.H. ; Eck, H.J. van; Ham, R.C.H.J. van; Jong, J.H.S.G.M. de; Visser, R.G.F. ; Bachem, C.W.B. - \ 2015
BMC Genomics 16 (2015). - ISSN 1471-2164
Background In flowering plants it has been shown that de novo genome assemblies of different species and genera show a significant drop in the proportion of alignable sequence. Within a plant species, however, it is assumed that different haplotypes of the same chromosome align well. In this paper we have compared three de novo assemblies of potato chromosome 5 and report on the sequence variation and the proportion of sequence that can be aligned. Results For the diploid potato clone RH89-039-16 (RH) we produced two linkage phase controlled and haplotype-specific assemblies of chromosome 5 based on BAC-by-BAC sequencing, which were aligned to each other and compared to the 52 Mb chromosome 5 reference sequence of the doubled monoploid clone DM 1–3 516 R44 (DM). We identified 17.0 Mb of non-redundant sequence scaffolds derived from euchromatic regions of RH and 38.4 Mb from the pericentromeric heterochromatin. For 32.7 Mb of the RH sequences the correct position and order on chromosome 5 was determined, using genetic markers, fluorescence in situ hybridisation and alignment to the DM reference genome. This ordered fraction of the RH sequences is situated in the euchromatic arms and in the heterochromatin borders. In the euchromatic regions, the sequence collinearity between the three chromosomal homologs is good, but interruption of collinearity occurs at nine gene clusters. Towards and into the heterochromatin borders, absence of collinearity due to structural variation was more extensive and was caused by hemizygous and poorly aligning regions of up to 450 kb in length. In the most central heterochromatin, a total of 22.7 Mb sequence from both RH haplotypes remained unordered. These RH sequences have very few syntenic regions and represent a non-alignable region between the RH and DM heterochromatin haplotypes of chromosome 5. Conclusions Our results show that among homologous potato chromosomes large regions are present with dramatic loss of sequence collinearity. This stresses the need for more de novo reference assemblies in order to capture genome diversity in this crop. The discovery of three highly diverged pericentric heterochromatin haplotypes within one species is a novelty in plant genome analysis. The possible origin and cytogenetic implication of this heterochromatin haplotype diversity are discussed.
Possibilities and challenges of the potato genome sequence
Visser, R.G.F. ; Bachem, C.W.B. ; Borm, T.J.A. ; Boer, J.M. de; Eck, H.J. van; Finkers, H.J. ; Linden, G. van der; Maliepaard, C.A. ; Uitdewilligen, J.G.A.M.L. ; Voorrips, R.E. ; Vos, P.G. ; Wolters, A.M.A. - \ 2014
Potato Research 57 (2014)3-4. - ISSN 0014-3065 - p. 327 - 330.
This paper describes the progress that has been made since the draft genome sequence of potato has been obtained and the analyses that need to be done to make further progress. Although sequencing has become less expensive and read lengths have increased, making optimal use of the information obtained is still difficult, certainly in the tetraploid potato crop. Major challenges in potato genomics are standardized genome assembly and haplotype analysis. Sequencing methods need to be improved further to achieve precision breeding. With the current new generation sequencing technology, the focus in potato breeding will shift from phenotype improvement to genotype improvement. In this respect, it is essential to realize that different alleles of the same gene can lead to different phenotypes depending on the genetic background and that there is significant epistatic interaction between different alleles. Genome-wide association studies will gain statistical power when binary single nucleotide polymorphism (SNP) data can be replaced with multi-allelic haplotype data. Binary SNP can be distributed across the many different alleles per locus or may be haplotype-specific, and potentially tag specific alleles which clearly differ in their contribution to a certain trait value. Assembling reads from the same linkage phase proved to allow constructing sufficiently long haplotype tracts to ensure their uniqueness. Combining large phenotyping data sets with modern approaches to sequencing and haplotype analysis and proper software will allow the efficiency of potato breeding to increase.
A Next-Generation Sequencing Method for Genotyping-by-Sequencing of Highly Heterozygous Autotetraploid Potato
Uitdewilligen, J.G.A.M.L. ; Wolters, A.M.A. ; hoop, B.B. D'; Borm, T.J.A. ; Visser, R.G.F. ; Eck, H.J. van - \ 2013
PLoS ONE 8 (2013)5. - ISSN 1932-6203 - 14 p.
single-nucleotide polymorphisms - genome-wide association - chloroplast dna - solanum-tuberosum - agronomic traits - hybrid selection - discovery - enrichment - resistance - diversity
Assessment of genomic DNA sequence variation and genotype calling in autotetraploids implies the ability to distinguish among five possible alternative allele copy number states. This study demonstrates the accuracy of genotyping-by-sequencing (GBS) of a large collection of autotetraploid potato cultivars using next-generation sequencing. It is still costly to reach sufficient read depths on a genome wide scale, across the cultivated gene pool. Therefore, we enriched cultivar-specific DNA sequencing libraries using an in-solution hybridisation method (SureSelect). This complexity reduction allowed to confine our study to 807 target genes distributed across the genomes of 83 tetraploid cultivars and one reference (DM 1–3 511). Indexed sequencing libraries were paired-end sequenced in 7 pools of 12 samples using Illumina HiSeq2000. After filtering and processing the raw sequence data, 12.4 Gigabases of high-quality sequence data was obtained, which mapped to 2.1 Mb of the potato reference genome, with a median average read depth of 63× per cultivar. We detected 129,156 sequence variants and genotyped the allele copy number of each variant for every cultivar. In this cultivar panel a variant density of 1 SNP/24 bp in exons and 1 SNP/15 bp in introns was obtained. The average minor allele frequency (MAF) of a variant was 0.14. Potato germplasm displayed a large number of relatively rare variants and/or haplotypes, with 61% of the variants having a MAF below 0.05. A very high average nucleotide diversity (p = 0.0107) was observed. Nucleotide diversity varied among potato chromosomes. Several genes under selection were identified. Genotyping-by-sequencing results, with allele copy number estimates, were validated with a KASP genotyping assay. This validation showed that read depths of ~60–80× can be used as a lower boundary for reliable assessment of allele copy number of sequence variants in autotetraploids. Genotypic data were associated with traits, and alleles strongly influencing maturity and flesh colour were identified.
Whole genome shotgun sequencing in a Solanum tuberosum interspecific backcross genotype
Xu, X. ; Pan, S.K. ; Cheng, S.F. ; Zhang, B. ; Bachem, Christian ; Boer, Jan de; Borm, Theo ; Kloosterman, Bjorn ; Eck, Herman van; Datema, Erwin ; Goverse, Aska ; Ham, Roeland van; Visser, Richard - \ 2011
Wageningen University and Research Centre
PRJEB2504 - PRJEB2504 - ERP000627 - Solanum tuberosum - Solanum phureja
Whole genome shotgun sequencing of hetrozygous diploid interspecific hybrid backcross (Solanum tuberosum ssp. phureja x Solanum tuberosum ssp. tuberosum) x Solanum tuberosum ssp. tuberosum) clone RH89-039-16.
A hybrid BAC physical map of potato: a framework for sequencing a heterozygous genome
Boer, J.M. de; Borm, T.J.A. ; Jesse, T. ; Brugmans, B.W. ; Tang, X. ; Bryan, G.J. ; Bakker, J. ; Eck, H.J. van; Visser, R.G.F. - \ 2011
BMC Genomics 12 (2011). - ISSN 1471-2164 - 60 p.
quantitative trait loci - candidate genes - disease resistance - linkage map - tomato - aflp - solanum - markers - dna - construction
Background Potato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH). Results First, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps in silico anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP) BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map. Conclusions The AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and ordering of genomic sequences of clone RH (and other potato genotypes), and opens the possibility to finish sequencing of the RH genome in a more efficient way via high throughput next generation approaches.
Genome sequence and analysis of the tuber crop potato
Xu, X. ; Pan, S.K. ; Cheng, S.F. ; Zhang, B. ; Bachem, C.W.B. ; Boer, J.M. de; Borm, T.J.A. ; Kloosterman, B.A. ; Eck, H.J. van; Datema, E. ; Goverse, A. ; Ham, R.C.H.J. van; Visser, R.G.F. - \ 2011
Nature 475 (2011). - ISSN 0028-0836 - p. 189 - 195.
eukaryotic genomes - resistance genes - in-vitro - rna-seq - solanum - tool - identification - elements - reveals - maize
Potato (Solanum tuberosum L.) is the world’s most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop
A genome-wide genetic map of NB-LRR disease resistance loci in potato
Bakker, E.H. ; Borm, T.J.A. ; Prins, J.C.P. ; Vossen, E.A.G. van der; Uenk, G.E. ; Arens, M.J.B. ; Boer, J.M. de; Eck, H.J. van; Muskens, M. ; Vossen, J. ; Linden, C.G. van der; Ham, R. van der; Klein Lankhorst, R.M. ; Visser, R.G.F. ; Bakker, J. ; Goverse, A. - \ 2011
Theoretical and Applied Genetics 123 (2011)2. - ISSN 0040-5752 - p. 493 - 508.
nucleotide-binding site - downy mildew resistance - late blight resistance - rich repeat class - race-specific resistance - flax rust resistance - nematode globodera-rostochiensis - tuberosum ssp andigena - tobacco-mosaic-virus - triticum-aestivum l.
Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato
|Cytogenetic distribution of chromosome 5 BAC sequences from the diploid potato genotype RH
Boer, J.M. de; Tang, X. ; Datema, E. ; Borm, T.J.A. ; Bakker, E.H. ; Lintel-Hekker, B. ; Ham, R.C.H.J. van; Jong, H. de; Bachem, C.W.B. ; Visser, R.G.F. - \ 2010
|An integrated genome wide genetic map of sequenced NBS-LRR disease resistance gene homologues (RGH) and resistance loci in potato
Bakker, E.H. ; Borm, T.J.A. ; Prins, J.C.P. ; Geest, A.H.M. van der; Vossen, E. van der; Uenk, G.E. ; Sabatino, G.J.H. ; Arens, M.J.B. ; Nap, J.P.H. ; Boer, J.M. de; Eck, H. van; Lintel Hekkert, B. te; Ham, R.C.H.J. van; Vossen, J. ; Linden, G. van der; Muskens, M. ; Allefs, S. ; Bakker, J. ; Goverse, A. - \ 2010
In: Potato breeding after the completion of the dna sequence of the potato genome, EUCARPIA - EAPR joint meeting, Wageningen, The Netherlands, 27 - 30 June, 2010. - Wageningen : Eucarpia - p. 53 - 53.
Construction and use of a physical map of potato
Borm, T.J.A. - \ 2008
Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Herman van Eck. - S.l. : S.n. - ISBN 9789085852377 - 139
solanum tuberosum - moleculaire kartering - genetische kartering - genomen - dna-fingerprinting - dna-bibliotheken - genetische merkers - maximale aannemelijkheid - moleculaire merkers - marker assisted breeding - aflp - solanum tuberosum - molecular mapping - genetic mapping - genomes - dna fingerprinting - dna libraries - genetic markers - maximum likelihood - molecular markers - marker assisted breeding - amplified fragment length polymorphism
Feeding the growing world population is one of the biggest challenges for the 21st century.
Potato, being the fourth crop in the human diet, after maize, wheat and rice, plays an
important role in this respect. Like other crops, potato is exposed to a range of potentially
yield-reducing factors: Pathogens, a (possibly changing) bad climate and averse soil
conditions. Research into the response of potato to these influences, often determined by
hereditary factors, is necessary to meet a growing demand for potatoes. A map of
genetically determined properties is crucial for this research. Several techniques are
available to produce maps – each with it's own merits and demerits, resulting in maps of
different qualities and with different resolutions. Two often used mapping techniques are
genetic mapping, where the inheritance of multiple traits (“markers”) is studied in
offspring using statistical analysis and the markers ordered accordingly, and physical
mapping on the basis of “Bacterial Artificial Chromosome” (BAC) libraries. BAC
libraries consist of a large number of individual bacterial strains (BAC clones), each
containing a randomly sampled section of DNA of the organism being studied. By
comparing individual BAC clones with each other, finding out where the donor organism's
(the organism being studied) DNA sections overlap, the BAC clones can be ordered into
groups or “contigs”. Comparison is often done on the basis of so called fingerprints – a
pattern consisting of DNA fragments of different lengths, resembling a bar-code pattern. A
similarity in fingerprint patterns between two BAC clones indicates that the BAC clones
contain similar (overlapping) sections of the donor organism's DNA. Recently an ultra
dense genetic map has been published, containing more than 10,000 markers produced
using “Amplified Fragment Length Polymorphism” (AFLPTM) marker technology. The
integrated physical and genetic map that is the subject of this thesis extends this genetic
map, and is in itself the starting point for determining the detailed DNA sequence of
potato, as is currently being undertaken by an international scientific collaboration within
the Potato Genome Sequencing Consortium (PGSC, http://www.potatogenome.net).
First step in creating this integrated physical and genetic map was creation, fingerprinting
and characterization of a BAC library, as described in chapter two. BACs were
individually fingerprinted using an AFLP based protocol, and (amongst others) these
AFLP BAC-fingerprints were compared to a theoretical model of the distribution of
fragment lengths in AFLP fingerprints to determine if fingerprinting was successful.
Correction and refinement of some of the mapping algorithms that were used to create the
genetic map are discussed in chapters three and four, resulting in refined genetic map
locations for the AFLP markers and the capability to process marker scores containing
arbitrary types of scoring ambiguities while conserving all available information. An
extension to the basic principle offers the possibility to also map AFLP markers derived
from different chromosomes that are indistinguishable on the basis of their AFLP
fragment length alone.
In chapter five, systematic differences in AFLP BAC fingerprints are discussed that are
caused by the use of different machines for capillary electrophoresis, by the use of
different fluorescent DNA labels and by different capillary position. These systematic
differences are (partially) corrected by using the (abundant) AFLP fingerprints of BAC
clones containing (part of) the potato chloroplast genome as a reference sample.
By ordering the AFLP fingerprints of individual BAC clones on the basis of fingerprint
similarity, a physical map is produced that is integrated with the genetic map using a
novel, ultra efficient, procedure described in chapter six. This procedure, “AFLP contig
matching” uses intricate experimental design and combinatorial analysis to obtain an
integrated physical and genetic map with the least amount of effort.
|Construction of a genetically anchored physical map of the potato genome
Boer, J.M. de; Borm, T.J.A. ; Brugmans, B.W. ; Werij, J.S. ; Wiggers-Perebolte, L. ; Jesse, T. ; Bakker, E.H. ; Keizer, L.C.P. ; Bakker, J. ; Visser, R.G.F. ; Eck, H.J. van - \ 2005
We are well underway with the construction of a genome-wide physical map of potato. For this goal a two-enzyme, 70,000 BAC library has been constructed of the heterozygous diploid genotype RH89-039-16. This library has an average insert size of over 120 kbp and has an expected coverage of 9-10 genome equivalents. The BACs have been fingerprinted by non-selective AFLP PCR and capillary electrophoresis on a MegaBACE sequencer. Physical map contigs will be constructed using two independent approaches. Firstly, BACs will be grouped into contigs using the computer program FPC (Soderlund et al., CABIOS 13:523-535). A draft first version of the physical map based on this analysis will be presented. Secondly, contigs will be identified using AFLP markers from the ultra dense genetic map of potato (http://potatodbase.dpw.wau.nl/UHDdata.html). In this second approach, selective (+3/+3 Eco/Mse) AFLP markers, made visible in fingerprints of BAC pools, will be compared with co-migrating non-selective (+0/+0 Eco/Mse) markers in the individual BAC fingerprints. In this way, an extensive integration of the physical map and the genetic map of potato will be achieved. This integrated map will serve as a source for map-based cloning of genes for disease resistance and tuber quality traits.