Foolad and Panthee (2012) give an excellent review of Marker
Assisted Selection (MAS) in tomato breeding, providing more detail than
intended here. Our goal is to explain
the basics and give examples of how this tool can be used by tomato breeders,
and how we are employing the technology in our breeding program.
There are various types of molecular markers that can be
used in breeding (e.g. SNP, AFLP, SCAR, CAPS and SSR), but these all represent
a short DNA sequence with a known physical location in the genome – essentially
“mile markers” on the roadmap that defines structural characteristics of the
plant DNA sequence. For example there
are tens of thousands of SNP markers now placed on the physical map of the tomato
genome. When one of these markers is
adjacent to (or within) a gene of interest, such as a gene associated with
resistance to a particular pathogen, the marker and the gene co-segregate – and
selection for the marker is an effective surrogate for selection for the
trait.
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| Distribution of SNP markers on tomato genetic map (Viquez-Zamora et al., 2013) |
PCR-based genotyping can be used to quickly confirm presence
or absence of a particular molecular marker, and many markers are co-dominant enabling a determination of whether the plant contains one (heterozygous) or
two (homozygous) copies of the marker.
These assays are conducted on DNA extracted from a very small amount of
plant tissue, and thus can be performed on very young seedlings.
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| Taking tomato tissue samples for molecular marker analysis |
Some of the advantages are obvious:
1)
The presence of the molecular marker can be
confirmed at the seedling stage, and not reliant on a trait phenotype that is
expressed several weeks later (e.g. fruit color), or on a phenotype that is
dependent on particular environmental conditions (e.g. disease resistance). Early selection allows early culling of undesirable
plants based on genotype rather than phenotype.
Early culling means that only those plants pre-selected for the desired
trait or combination of traits go to field breeding nurseries or crossing
programs.
2)
Trait stacking is the breeding process for
combining multiple desirable traits into a single breeding line. The first step always involves using crossing
to bring the multiple traits into a single breeding population. The second step, greatly aided by MAS, is to
identify low frequency plants in the breeding population that contain all the
traits of interest. For example: the
cross AABBCC x aabbcc, where genes A and B control resistance to two independent
tomato pathogens (e.g. ToMV and LB) and resistance is dominant, and c is a
desirable recessive allele for a trait for fruit color (e.g. ogc/crimson). The F1 progeny will all be AaBbCc and in the
F2 only one plant in 64 will have the desired stable genotype AABBcc. Now think about stacking 7-8 genes (which we are) - MAS allows the efficient testing
of hundreds of F2 progeny to find the “needles in the haystack” combing the
desired traits. When stacking more than 3 traits it will likely be necessary to do this in a stepwise fashion.
How is MAS being used today?
Molecular markers have now been identified for many of the multiple genes
associated with resistance to key tomato diseases and nematodes, and in some
cases to the multiple races of the diseases now prevalent (e.g, all three races
of Fusarium wilt). Commercial breeders
have been successful at stacking resistance to most of these key pathogens in
newer hybrids – with MAS being a critical tool in such stacking.
Molecular markers have also been identified for a handful of other major
genes controlling important traits – but it is a short list: tomato fruit color - red vs yellow and
crimson vs red; fruit size - locule number; plant type - determinant vs indeterminant; and presence of the rin allele
associated with delayed ripening. Some of the major genes controlling plant/fruit phenotype are shown below (photo courtesy of University of Newfoundland) - linked molecular markers are available for a few of these.
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| Map of major genes controlling plant/fruit phenotype |
All of the examples cited above involve major genes providing control of
simply inherited traits. However we know
that many important traits are quantitative traits, controlled by multiple
genes – typically each with a small, but cumulative effect. Quantitative trait loci (QTL) are molecular
markers associated with genes/alleles contributing to a quantitative trait –
such a fruit yield, fruit size and fruit flavor. MAS allows the effective stacking of QTLs
that in concert have a major impact on a “hard to breed for” quantitative
traits.
Harry Klee and his colleagues at the University of Florida have been
working to unlock the mystery of flavor in tomatoes. What are the multiple
components of flavor, how do they interact, and what is the genetic control of
these factors? I am confident this will
eventually lead to QTLs associated with key flavor components – facilitating
breeding for better tasting tomatoes.
University breeding programs and the larger commercial tomato breeding
companies all have access to the tools required for MAS. Thankfully there are also a few commercial
companies that conduct such activities on a fee for service basis, which allows
the smaller players (FLF included) access to these tools. Such access is a significant incremental
expense per se, but allows cost savings, and an accelerated timeline in the
long run.
Our
breeding program started with crosses between heirloom types with a primary
goal to improve flavor and plant health.
After a couple years we started crossing these to a handful of
commercial types, including a couple of the NCSU hybrids, to introduce improved
disease tolerance and improved fruit quality.
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| Disease resistant X Great Flavor |
We are now using molecular markers to identify progeny from these
crosses which combine resistance to multiple pathogens – and will follow that genotypic
selection with phenotypic selection for flavor and fruit quality in our various
breeding nurseries. The use of molecular
markers and our recent access to facilities allowing 3 breeding generations/yr
should allow us to soon commercialize new F1 hybrids with state-of-the-art disease
resistance, best-in-class heirloom flavor, and in a rainbow’s array of colors
and stripes.
MAS-enabled multiple disease resistant F4 - a F1 parent "in training"
