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.