Introduction

Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA

sequences that can be detected by the presence of fragments of different lengths after digestion
of the DNA samples in question with specificrestriction endonucleases. RFLP, as a molecular
marker, is specific to a single clone/restriction enzyme combination.

Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and
highly locus-specific.

An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the
digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique
blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy
genomic DNA or cDNA clones are typically used as RFLP probes.

The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping,
forensics, paternity tests, hereditary disease diagnostics, etc.).

The bacterial strains used in this study are shown in Table 6-1. These strains
were isolated as plant pathogens, identified as X. campestris, and classified into
pathovars according to the susceptible host plants involved. Strains were tested
for pathogenicity after single colony purification. For some strains, no pathovar
assignments were made due to the lack of known plant pathogenic responses.
Broth cultures of bacteria were grown in a peptone-glycerol medium (per L:
10.0 ml glycerol, 20.0 g peptone, and 1.5 g K2HPO4; pH 7.2). The strains were
commonly stored and maintained at -80°C in the same medium containing 15%
glycerol.

Discussion
The only widely accepted and most practical method for differentiating
pathovars of X. campestris is to inoculate a plant suspected as the host for that
pathovar. This practice can sometimes be tedious, time consuming, subjective
and subject to a surprising number of artifactual influences. It is not known
whether host selectivity is unstable as suggested by Dye [358]; stable as
suggested by Schnathorst [359] or even taxonomically significant. Although the
classification is thought to be useful, it can be highly misleading since
emphasis is placed upon one characteristic - pathogenicity. If only one or few
gene differences were involved in host selection, then differentiation by
pathovar could be highly misleading, at least in the sense that a given group of
strains might be capable of attacking more than one host in some cases.
Alternatively, two or more strains of relatively unrelated groups could be
cataloged together because they happen to attack the same host. This latter
situation appears to be the case with X. campestris pv. phaseoli and X.
campestris pv. phaseoli var. fuscans (see Fig. 6-3), and with Florida strains
of X. campestris pv. citri [276].

In order to address these problems, we evaluated the potential of using DNA

sequence variation of X. campestris for strain classification purposes. DNA
sequence variation was first examined by digesting DNA with restriction
enzymes and visualizing directly the resulting fragments on ethidium bromide
stained gels. These stained gels were useful for side by side comparisons of
restriction fragments for samples run on the same gel, but comparisons between
different gels were more difficult. Subsequently, visualizing and comparing
variation in bacterial genomes with cosmid clones carrying 30-38 kb cloned X.
campestrisgenome fragments was simplified. In both cases variation was
revealed by alterations in the sizes of visualized DNA restriction fragments
(RFLPs). The use of DNA probes derived from chromosomal DNA fragments
also alleviated some of the difficulty in comparing stained gels with the
sometimes present polymorphism of the higher copy number plasmid DNA
fragments which can occur in the background.

Some of the selected clones tested as DNA probes appeared to be useful for
identifying DNA sequences conserved within a given pathovar, while others
appeared to identify DNA sequences which were highly conserved at the
species level. The DNA which was considered conserved at the species level
was represented as banding patterns which were nearly identical among all
strains over the several pathovars tested. Examples of DNA sequences which
are known to be highly conserved are rRNA encoding genes [212,213]. There
are undoubtedly others. If smaller DNA probes containing known genetic loci
such as those associated with genes for pectate lyase and protease [29], or
avirulence activity [106,234] were tested, these smaller, more defined DNA
probes could be used to determine if such genetic loci were highly conserved or
variable. With larger, randomly selected probes, the likelihood was increased
for detecting strain- or pathovar-specific variation. The large (greater than 30
kb) size of the probes used in this study allowed detection of both.

Using RFLP genomic blots, it appeared that phylogenetic relationships between

the full spectrum of described pathovars of X. campestris might be
determinable. Some mathematical approaches toward determining phylogeny
based on restriction cleavage sites has been proposed [342,346]. DNA probes
have been used to make phylogenetic comparisons among the relatively
conserved mitochondrial [320] and chloroplast [349] genomes. The variation
seen among the different pathovars of X. campestris appeared within the range
that is usefully distinguishable with this test. Only limited amounts of variation
can be usefully distinguished, such as that occurring within a species.
Significantly, the RFLP groupings closely corresponded with the pathovar
groupings, strengthening the taxonomic significance of this classification. All
strains tested were readily grouped by RFLP phenotypes, and the classification
based on RFLP patterns correlated very well with the classification based on
pathogenicity. This technique may provide a more convenient means of
classifying these bacteria. In addition, unexpected taxonomic relationships
between pathovars might be revealed. The potential pathogenic range of each
RFLP group would be of obvious value.

By comparing observed RFLPs among strains of X. campestris using selected

DNA probes, it was possible to identify unknown strains when known
standards were included. Additionally, strains previously undescribed could be
classified as being related to known pathovars. For instance, similarities were
found between undescribed strains isolated from a Alocasia sp. and Argemone
sp.; and a Cissus sp. and Jasmine specie. The strains isolated from a
Philodendron sp. and Dieffenbachia sp. both corresponded to that of X.
campestris pv. dieffenbachiae. The other unknown strains tested appeared
different from one another, and did not appear similar to any of the other X.
campestris pathovars as characterized by the DNA probes. Finally, this
technique may provide a basis for the classification of nonpathogenic and
epiphytic xanthomonads. Experiments can now be conducted to determine
whether epiphytes are all basically similar or form diverse groups, whether they
are restricted in host range or if they are nonpathogenic, but closely related to
described pathovars. Although this approach provides another classification
scheme for strains, it appears to be basically compatible with the currently
recognized and useful pathovar naming scheme, which relies primarily on
plant-specificity. Although certain pathovars may need to be redefined, this
work supports and helps validate the natural taxonomic groupings provided by
the pathovar naming system.

Result
The distance between the locations cut by restriction enzymes (the restriction sites) varies
between individuals: so the length of the fragments varies, and the position of certain gel
bandsdiffers between individuals (thus polymorphism). This can be used to genetically tell
individuals apart. It can also show the genetic relationship between individuals, because
children inherit genetic elements from their parents. It is also used to deterimne the
relationships among species.

RESULTS AND DISCUSSION

Our DNA isolation technique was developed from a previous method based on
cell lysis in hypotonic, non-ionic detergent, low-speed centrifugation to pellet
nuclei, disruption of nuclei, and partial proteinase TOP
K digestion in a low ionic strength buffer (Ding, ABSTRACT
INTRODUCTION
1992). We modified this technique for use in 96- MATERIALS AND METHODS
well format and used pronase E, a protease that RESULTS AND DISCUSSION
can be denatured at 65°C, thus maintaining the REFERENCES
native, double-strand form of the DNA. We
evaluated the DNA yield from fresh or frozen chicken blood, chicken
semen, chicken feather pulp, and bovine blood (Table 1 ). As can be seen from
the data in Table 1 , there is a nearly linear relationship between isolated DNA
concentration and chicken blood volume using our procedure. The quantity of
DNA is sufficient for hundreds of standard PCR genotype analyses (e.g.,
microsatellite and single nucleotide polymorphism analysis).

View this table: Table 1. Yield of DNA from a variety of sources1

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Our procedure yields intact DNA sufficiently pure for restriction endonuclease
treatment. We compared DNA isolated from chicken blood by our method to
DNA purified using standard SDS lysis, proteinase K digestion, organic
extraction, and ethanol precipitation (Grimberg et al., 1989). The DNA aliquots
were digested with various enzymes (AluI, BamHI, EcoRI, or HindIII).
Electrophoretic patterns from digested DNA were indistinguishable between
the DNA isolated by the 2 different methods (data not shown). Southern blot
analysis using a cDNA probe to ER (provided by D. Bunik, University of
Illinois, Urbana-Champaign) showed no differences between the hybridization
patterns (Figure 1 ). Thus, this method inactivates nonspecific nucleases,
introduces no inhibitorycompounds, and produces DNA solutions suitable for
restriction endonuclease digestion for Southern blot analysis and RFLP
genotyping.

Figure 1. Comparison of DNA restrictability: Southern blot

of HindIII digested DNA samples (2 µg/lane) probed with a
cDNA for estrogen receptor . Lanes 1 to 3: DNA samples
prepared by our method; lanes 4 to 5: DNA samples purified by
standard methods. Molecular weights based on DNA standards
are indicated to the right of the figure.

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To evaluate the DNA isolation procedure for high-throughput PCR genotyping,

we isolated DNA from 886 frozen blood samples archived for over 1 yr in a
frost-free freezer using our toothpick method. Typically, blood samples stored
in this manner can be of poor quality (cell lysis) or be difficult to pipet when
thawed (clots). After DNA isolation, quantification of 25 random
samples showed a variable range of DNA concentrations (Table 1 ).
Given these concentrations, we used 2 µL of the DNA solution in 20-µL PCR
amplifications for genotyping at 2 microsatellite loci, ADR001 and KS001. For
the 886 archived chicken blood samples, we obtained genotype data for 792
samples for ADR001 and 737 samples for KS001, where 78 samples amplified
for ADR001 only, 28 samples amplified for KS001 only, and 68 samples gave
no amplification for either locus. The latter are most likely because of low DNA
quantities for those samples or pipetting errors. Therefore, using our rapid DNA
isolation method, our PCR success rate was 818 out of 886 or 92.3%, based on
the ability to genotype at least 1 locus for each DNA sample.

We have used DNA extracted by our method from semen for hundreds of
microsatellite PCR genotype determinations (data not shown). We compared
DNA isolated by our method from single feathers to DNA extracted using
alkaline treatment (Rudbeck and Dissing, 1998; Malago et al., 2002). Figure 2
shows PCR data comparing these DNA samples when amplified with WXho
and Ribo sexing primers. Only 9 of the 24 DNA samples isolated by NaOH
extraction yielded adequate amplification, whereas all 24 of the samples
isolated by TEN+pronase digestion amplified, with 1 DNA sample
producing only weak amplification, and 2 amplifying only the W-
specific fragment. We also tested these DNA samples for
microsatellite genotyping with ADR001 and KS001 and found that 9 of 10
TEN+pronase DNA samples amplified for both loci whereas zero of 10 NaOH-
extracted DNA samples amplified for either locus (Figure 3 ). We tested these
same DNA samples for 2 additional microsatellite loci (LEI0217 and
UMA1.070) and produced amplifications for 3 of 10 NaOH-extracted DNA
samples vs. 10 of 10 TEN+pronase-extracted DNA samples (data not shown).
Therefore, in our experience, the TEN+pronase digestion is much more reliable
and produces DNA more amenable to PCR genotyping.

Figure 2. Polymerase chain reaction sexing of DNA

samples isolated by alkaline extraction or by
pronase digestion. The DNA samples were
subjected to PCR using the WXho and Ribo
primers, and resolved in a 1.5% agarose gel. Top:
24 DNA samples extracted from feather pulp by the
NaOH method from different hens. Bottom: DNA
samples extracted from feather pulp by
TEN+pronase (Tris-EDTA-NaCl + pronase)
digestion from the same hens as in the top panel.
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Figure 3. Microsatellite PCR analysis of DNA

samples isolated by alkaline extraction or by
pronase digestion. The DNA samples were
amplified for ADR001 (upper panel) and KS001
View larger version (47K): (lower panel). Lanes 1 to 10: DNA extracted from
[in this window] feather pulp by NaOH treatment; lane 11: DNA
[in a new window] purified from blood by standard methods; lanes 12
to 21: DNA extracted from feather pulp by
TEN+pronase (Tris-EDTA-NaCl + pronase)
digestion; lanes 22 and 23: no input DNA; lane M:
molecular weight markers. Lengths (bp) of marker
bands are indicated to the right.

We adapted the procedure to work with mammalian blood samples. Based on

the low frequency of nucleated cells in mammalian whole blood and greater
starting sample volumes, we found that 2 successive STM treatments were
required to reduce contaminating proteins and avoid coagulated proteins upon
enzyme denaturation at 65°C. When we used 2 successive centrifugation steps
we obtained final DNA that could be successfully amplified in PCR using
primers to bovine HSP70 (provided by C. Rosenkrans, University of
Arkansas, Fayetteville; data not shown).

This simple, and inexpensive method for isolation of DNA from a variety of
sources will facilitate the rapid, high-throughput PCR or RFLP analysis of
genotypes in avian and other species in situations where overall costs must be
minimized.