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View the complete scientific
paper: Genetic Diversity among U.S. Garlic Clones
as Detected Using AFLP
Methods (381 KB Adobe Acrobat file)
GAYLE VOLK, ADAM HENK & CHRISTOPHER
RICHARDS / USDA-ARS National Center for Genetic Resources
Preservation Ft. Collins, Colorado
We have recently finished a genetic analysis of 211 Allium
sativum and Allium longicuspis accessions from commercial
and USDA sources. We know that garlic, in general, tends to
be very responsive to the environment and cultivars that thrive
in some locations can do very poorly at others. We suspect
that these different responses are dependent upon soil type,
moisture, latitude, altitude, and cultural practices. We also
know that garlic varieties have been renamed multiple times
as they have been passed between growers and gardeners. As
a result, many varieties may be identical genetically, yet
have unique names. We used a fingerprinting method called
AFLP (Amplified Fragment Length Polymorphism) to compare the
DNA of different garlic cultivars. A general description of
this method can be found at this website. Using the AFLP method,
we have identified many identical as well as numerous unique
garlic accessions in federal and commercial collections. The
studies presented in this summary will soon be published in
the Journal of the American Society for Horticultural Science.
Garlic is botanically known as Allium sativum. Another described
species, A. longicuspis, can be found in the wild in Central
Asia and was once thought to be the living progenitor of A.
sativum. USDA's National Plant Germplasm System (NPGS) maintains
193 main accessions of garlic at the Western Regional Plant
Introduction Station (WRPIS) in Pullman, WA. One hundred eighteen
of these accessions were provided by Barbara Hellier (collection
curator) and included in our current study. There are many
additional named garlic varieties that are available through
growers nationwide. We included 75 commercially available
varieties that were generously provided by Walt Lyons (www.thegarlicstore.com)
and Tom Cloud (www.filareefarms.com).
In our dataset we included some accessions that had the same
names but were obtained from different sources. Accessions
duplicated in this manner were identified in tables and figures
with an appended number on the name, for example 'Siberian-1'
and 'Siberian-2'. We included these duplications to test if
clones bearing the same name from two sources were genetically
similar.
Genetic Methods
A description of our methods is detailed in our full-length
publication. We briefly describe our methods and techniques
in this summary. We extracted DNA from two tissue samples
from garlic shoot tips within cloves from each of the 211
accessions, which gave us 422 samples. We then followed the
standard AFLP protocol as described by Vos et al. (1995).
After a series of treatments and digesting the DNA with enzymes
that cut it into pieces, we had DNA fragments that we could
separate by size on a large, thin gel. In Figure 1, DNA fingerprints
of several garlic accessions are shown. One accession is represented
by a column of bands. Banding patterns differed among samples.
The rows of bands in this gel are all the same length. For
some pairs of accessions, the band might be absent (white
space), and in others, it may be present (black band). We
selected 27 rows of bands (each row is called a locus- a position
in the garlic genome) that we scored for all 422 samples.
We felt confident scoring 27 rows of bands that were variable
between samples, yet unambiguously black or white for all
the samples. We determined whether a band was present or absent
for each of the 27 loci in all the samples. We then made sure
that the fingerprint assigned was identical for both of the
two replicate DNA samples for a given garlic cultivar. For
158 samples, the scores were identical between the replicate
samples for each cultivar. For the remaining 53 of the 211
cultivars, 26 of the 27 loci scores were in agreement between
the two replicates. We considered that one locus to be a "missing
data point". We performed a number of statistical analyses
on these data to determine differences and similarities across
cultivars in our dataset. These results will be presented
in a detailed manner in our published paper. Here we concentrate
on the results revealed by one of our figures.
Results
We used the technique called a minimum spanning network that
uses genetic distance to graphically illustrate genetic diversity
among the complete set of 211 accessions (Figure 2). Genetic
distance measures how closely related one individual clone
is to another. This diagram shows similarity among the 211
accessions in our study. The length of the lines between nodes
in Figure 2 reflects genetic distance. In some cases, connections
among nodes create loops in the network where there are several
"closest" relatives. In fact, the network is better thought
of as a mobile in three dimensions that has been laid flat.
This would explain why lines connecting nodes that appear
far apart are linked in the two dimensional representation.
The large ellipse in the center of the network represents
accessions with many connections and appears to be the basal
or most primitive group of accessions because of its similarity
with an unrelated Allium species. The diameter of each node
is proportional to the number of accessions in that group.
We were able to identify garlic accessions that were unique,
and the names of those accessions are written directly on
Figure 2. Many of the clones, however, were clustered into
genetically identical groups listed as lettered nodes in the
network. The names of the clones that were assigned to a lettered
node are listed in Table 1.
Overall, 64% of the WRPIS and 41% of the commercial accessions
had some degree of duplication according to this method. Cultivars
may differ at loci that we did not examine. Therefore, cultivars
that we identified as belonging to the same group are genetically
very similar (but statistically identical). We certainly DO
NOT promote the renaming of known accessions since there could
be genetic differences among cultivars that were not identified
using our method.
Growers looking to maximize the diversity of accessions they
are growing should select a clone from among nodes in the
networks. Alternatively, if some growers know that accessions
listed under group "F" and "G" grew and sell particularly
well in their region, they may want to try other accessions
that are assigned to those nodes. Finally, if planting stock
for an accession listed under group "I" is unavailable, a
grower could try to grow a differently named, yet genetically
similar accession listed in the same group. Our genetic analysis
confirms previous observations of Pooler and Simon (1993),
Ipek et al. (2003), and Al-Zahim et al. (1997) that A. longicuspis
is indistinguishable from A. sativum and may not be an appropriate
taxonomic entity. These data also provide some indication
of the diversity of garlic lineages. There is substantial
structure in the network that indicates the genetic origins
of certain plant morphologies. An example of this is the split
between hardneck (dark nodes) and softneck (white nodes) clones.
These data suggest that the softneck neck type may have arisen
out of a much broader hardneck pool of diversity. These kinds
of analyses provide a framework for further studies on the
domestication process but also for the development of forensic
tools that can be used to provide the genetic identity of
garlic clones.
References
Al-Zahim, H.J. Newbury and B.V. Ford-Lloyd. 1997. Classification
of genetic variation in garlic (Allium sativum L.) revealed
by RAPD. HortScience. 32:1102-1104.
Ipek, M., A. Ipek and P.W. Simon. 2003. Comparison of AFLPs,
RAPD markers, and isozymes for diversity assessment of garlic
and detection of putative duplicates in germplasm collections.
J. Amer. Soc. Hort. Sci. 128:246-252.
Pooler, M.R. and P.W. Simon. 1993. Characterization and classification
of isozyme and morphological variation in a diverse collection
of garlic clones. Euphytica 68:121-130.
Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van de Lee,
M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper and
M. Zabeau. 1995. AFLP: A new technique for DNA fingerprinting.
Nucleic Acids Res. 23:4407-4414.
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