128 C.D. Clegg et al. Applied Soil Ecology 14 2000 125–134
culated using a bootstrap procedure Griffiths et al., 1996. If the target and probe DNA have the same
genetic composition then the reciprocal cross of the target-probe hybridisation will give the same S value.
If one DNA sample is more diverse i.e. has a greater range of genetic sequence types than the other, then
S is not equal and the pair of values are asymmetric Lee and Fuhrman, 1990; Griffiths et al., 1996. The
probe giving the highest S value is the more diverse of the two samples and the ‘true’ degree of similar-
ity is denoted by the lower of the two hybridisation values Lee and Fuhrman, 1990; Ritz and Griffiths,
1994.
The assay was carried out using two classes of DNA sample. One class involved the cross hybridis-
ation of DNA extracted from the individual quadrats within each vegetation type at each site; this tested for
the degree of similarity in community DNA between quadrats within vegetation types. A second class tested
Table 2 Yields and purities of DNA extracted from grassland soils
Site Grassland
Quadrat Yield mg g
− 1
dry soil Abs. ratio 260:280
Abs. ratio 260:230 Garrigill
Improved 1
6.30 1.52
1.76 MG6
2 13.6
1.32 2.02
3 35.7
1.35 2.21
Semi-improved 1
35.7 1.31
1.85 U4b
2 17.1
1.48 2.02
3 21.2
1.46 2.08
Unimproved 1
29.0 1.38
1.96 U4a
2 33.6
1.35 2.13
3 47.5
1.42 2.01
Aber Improved
1 19.4
1.71 1.60
MG6 2
27.3 1.66
1.50 3
23.8 1.85
1.74 Semi-improved
1 32.8
1.61 1.29
U4b 2
26.9 1.38
1.11 3
14.7 1.58
1.16 Unimproved
1 44.1
1.25 0.96
U4a 2
34.9 1.51
1.12 3
45.9 1.66
1.28 Sourhope
Improved 1
20.23 1.98
1.78 MG6
2 19.68
1.96 1.78
3 30.10
1.74 1.50
Semi-improved 1
57.42 1.53
1.19 U4b
2 69.82
1.31 1.06
3 25.50
1.66 1.20
Unimproved 1
14.96 1.74
1.44 U4a
2 19.58
1.58 1.58
3 49.84
1.60 1.23
for similarity between vegetation types within sites, and was based on aliquots of DNA produced by pool-
ing DNA from each of the quadrats within a vegeta- tion type.
2.5. Statistics Results of the various analyses were considered sta-
tistically significant at p0.05.
3. Results
3.1. Yield and purity of DNA from the soils Yields and purities of DNA extracted from the
soils are given in Table 2. Yields of DNA from both replicate quadrats and the different grasslands were
variable. Although average DNA yields from repli-
C.D. Clegg et al. Applied Soil Ecology 14 2000 125–134 129
cate quadrats were least for the improved grasslands, there was no significant difference in DNA yield
between vegetation sequences at Garrigill and also at Sourhope. At Aber however, the mean yield of
DNA was significantly greater from the unimproved grassland than from the semi-improved and improved
grasslands.
3.2. Thermal denaturation of DNA The G+C distribution profiles for the three grass-
lands at Garrigill are shown in Fig. 1. The G+C distribution profile for the improved grassland at Gar-
rigill Fig. 1 informs us that 9 of the total ex- tracted DNA was a G+C content of about 58. Sim-
ilarly, Fig. 1 also illustrates that 6 of the unim- proved DNA at Garrigill was about 49 G+C and
5.8 of the semi-improved DNA was about a 51 G+C content. Melting profile parameters were anal-
ysed by factorial ANOVA with an increasing hierar- chy of replicate melts per DNA sample. This enabled
the analysis of replicate quadrats within each grass- land type, grassland types at a single site, and dif-
ferent sites. Lowest error mean square EMS values were always obtained for replicate melts, but there
was no distinct hierarchy in EMS for the other fac- tors. In some instances there was as much variation
between quadrats as between grassland types and be- tween sites. The G+C distribution profiles of repli-
cate quadrats from each of the three grassland types at
Fig. 1. The G+C distribution profiles for unimproved · · · -, semi-improved – – and improved — grasslands at Garrigill.
Fig. 2. The G+C distribution profiles for replicate quadrats within a unimproved; b semi-improved; and c improved grass-
lands at Garrigill.
Garrigill are given in Fig. 2. Significant differences of curve parameters in grasslands were only detected be-
tween sites, and not within sites, in a combined anal- ysis Table 3. Within-site analysis indicated signifi-
cant differences in the DNA melting curve parame- ters between replicate quadrats within some individual
grasslands Table 3. Within-site analysis of the pa- rameters at the vegetation sequence level also revealed
that at Garrigill parameters b and m were significantly greater for the improved than the semi-improved and
unimproved grasslands. An increase in the value of b is indicative of a general narrowing of the range
of G+C distribution, and an increase in the value of m is indicative of an increase in median G+C
content in the community. Thus at Garrigill, DNA from the improved grassland generally contained a
higher proportion of DNA with a higher G+C con- tent than the DNA extracted from the semi-improved
130 C.D. Clegg et al. Applied Soil Ecology 14 2000 125–134
Table 3 Melting curve parameters and median G+C composition for the
vegetation sequences at Garrigill, Aber and Sourhope
a
Site Grassland
b m
t G+C
Garrigill Improved
0.66 81.0
1.46 62.3
Semi-improved 0.51
78.6
∗
1.44 57.9
Unimproved 0.52
77.5
∗
1.24 55.3
Aber Improved
0.45 78.4
1.83 57.3
Semi-improved 0.51
79.8
∗
1.53 59.9
Unimproved 0.51
∗
78.3
∗
2.93
∗
56.9 Sourhope
Improved 0.65
79.8 1.86
59.9 Semi-improved
0.57
∗
78.8
∗
1.73
∗
58.0 Unimproved
0.67 80.0
2.47 60.4
LSD
b
0.20 3.79
1.28
a
LSD values obtained from the combined analysis of all three sites.
b
LSD: least significant difference.
∗
Significant differences between replicate quadrats within grassland type.
and unimproved grasslands. No significant differences in the DNA melting curve parameters were detected
between the grasslands within the sites at Aber and Sourhope.
3.3. Cross hybridisation of DNA Hybridisation couplets were generally asymmetric,
i.e. when probe and target were switched the S values were significantly different. This permits the deter-
mination of which sample was the more complex in terms of DNA species content. When the sample used
as the probe gives the higher S value in the hybridisa- tion couplet, this is indicative that its DNA is the more
complex of the pair, whilst the lower index reflects the relative similarity between the samples. Thus the data
are summarised in Figs. 3 and 4 by showing the lowest value of the hybridisation couplets and the ranking of
complexity established on the basis described earlier. For pooled DNA samples, representing the average
community DNA within each grassland type, the de- gree of similarity was around 80 for all three vegeta-
tion types at Garrigill and Sourhope Fig. 3. However at Aber, greater variation between grassland types
was evident, with unimproved and semi-improved types only showing around 50 similarity Fig. 3.
There was also a general trend at the Garrigill
Fig. 3. Percentage similarity indices S, with 95 confidence in- tervals in brackets, for cross hybridisations of DNA extracted from
unimproved, semi-improved and improved vegetation sequences at a Garrigill; b Aber; and c Sourhope.
and Aber sites of relative complexity following the sequence;
unimprovedsemi-improvedimproved. However, at Sourhope the microbial community
from the improved grassland was more com- plex than that from the semi-improved grassland
Fig. 3. Cross hybridisation data for individual quadrats within grasslands at each site also had vary-
ing degrees of similarity, ranging from as low as 17 and up to 70 at Aber Fig. 4. Such variation
was also observed at Garrigill range 58–98; data not shown and Sourhope range 17–100; data not
shown.
C.D. Clegg et al. Applied Soil Ecology 14 2000 125–134 131
Fig. 4. Percentage similarity indices S, with 95 confidence intervals in brackets, for cross hybridisations of DNA extracted
from replicate quadrats within a unimproved; b semi-improved; and c improved vegetation sequences at the Aber site.
4. Discussion
