Construction and use of a physical map of potato
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.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Subjects: | amplified fragment length polymorphism, dna fingerprinting, dna libraries, genetic mapping, genetic markers, genomes, marker assisted breeding, maximum likelihood, molecular mapping, molecular markers, solanum tuberosum, aflp, dna-bibliotheken, dna-fingerprinting, genetische kartering, genetische merkers, genomen, maximale aannemelijkheid, moleculaire kartering, moleculaire merkers, |
| Online Access: | https://research.wur.nl/en/publications/construction-and-use-of-a-physical-map-of-potato |
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| Summary: | 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. |
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