Study of Populus deltoides genetic...
Transcript of Study of Populus deltoides genetic...
Genetic diversity 83
TERI School-Ph.D Thesis, 2004
Study of Populus deltoides genetic
diversity
4.1 Introduction
In India, six species of poplars are found distributed in the Himalayas: P. alba L.,
P. ciliata Royle, P. euphratica Oliv., P. gamblei Haines, P. glauca Haines and
P. suaveolens Fisch. (Khurana 2002). Besides these, India is rich in exotic genetic
resources of poplars (Review of Literature, Section 2.1.6). The exotic poplars have
been grown in many parts of the country as early as 1950, when Euro-American
clones were introduced from the U.K. and many other clones from Italy,
Germany, the Netherlands, the USA and Australia followed (Mathur and Sharma,
1983). However, poplar cultivation in India changed with the introduction of fast-
growing clones of Populus deltoides Marsh. from the USA and Australia.
The large number of P. deltoides clones introduced since 1950 onwards
have been screened for growth and yield in Indian conditions, and suitable clones
have been identified for mass propagation. After a trial of about 20 years,
covering about 100 clones of different species, certain clones were identified as
promising for field plantation. These trials, however, gave a clear indication that
the clones lost vigour and resistance to pests and diseases by successive
multiplication. It was thus realized that there needs to be a proper breeding
program for producing new promising clones in this species (Chaturvedi and
Rawat, 1994).
In 1982 and 1984, there was flowering in female P. deltoides G-48 clones
and they were open pollinated by G-3 clones in Dhimri block of Tarai Central
Forest Division (Lat. 29°10’ N, Long.79°40’E, Alt. 256m). The seedlings were
raised and planted in LalKuan Forest Reseach Nursery under the name of ‘L-
Series’. After a trial of about 8 years, seven clones out of 60 were identified as
better performing in the 1982 selections (Chaturvedi and Rawat, 1994).
Similarily, from the 1984 selections, 25 out of 113 clones were identified as
promising (Chaturvedi and Rawat, 1994). Trials in different locations and
climatic conditions have shown that P. deltoides clones perform well in all the
five tested sites which fall into. 1. Humid sub-temperate; 2. Riverian sub-
temperate; 3. Temperate; 4. Sub-tropical; and 5. Sub-humid sub-tropical zones
(Saraswat et al. 1993). Introduction of more clones from different parts of the
world is still continuing, and there is an ever increasing collection of P. deltoides
4
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
germplasm in the country today (Khurana and Narkhede 1995), resulting in a
need for proper cataloguing. The fundamental problems confronting poplar
breeders in India are the lack of information on the genetic diversity within and
between Populus species and accurate identification of clones. This knowledge is
essential for planning effective hybridization programmes, varietal control,
protection and registration of plant breeder’s rights, and day-to-day planning and
handling of breeding stocks. With the continuous import of P. deltoides clones in
India, there is an urgent need of preparation of proper database of this exotic
species. The objective of the present study was to determine the levels of intra-
specific genetic diversity in P. deltoides clones grown in India. AFLP markers
were utilized for assessing the diversity at molecular level.
Leaf material of the P. deltoides clones grown extensively in India were
collected for the present study. The source from where these clones were
obtained for introduction in India was identified for most of the clones except few
for which the original source was not known. Leaf samples were collected from
forty-three P. deltoides genotypes and three species viz. P. ciliata, P.
maximowiczii and P. euphratica genotypes were included as outliers in the
study. P. deltoides and P. ciliata leaf material was collected from Dr. YS Parmar
University of Horticulture and forestry, Nauni (Solan). The P. euphratica leaf
material was collected from TERI field-station, Gual Pahari, Gurgaon; and
P. maximowiczii leaf material was kindly provided by Dr. Stefano Bissoffi of
Poplar Research Institute, Italy. Table 4.1 shows the list of accessions analyzed
for genetic diversity studies using AFLP technique. The 43 better performing
P. deltoides clones were chosen for the present study. Sixteen of these clones are
introductions from the U.S.A. (clones M–Z, l and m), six from Netherlands (a–f),
three from Germany (h–j), and three from Australia (K, L and q) [Table 4.1]. Ten
clones (A–J) have been developed and selected in India, and for the remaining
five, the source of introduction was not known [Table 4.1]. The source of
introduction and origin of the clones and the code given for labeling them on
AFLP gels are also given in Table 4.1.
AFLP analysis was carried out using ten different primer combinations
that are listed in Table 4.2. AFLP protocol is described in Material and Methods,
Section 3.2.2. AFLP data was used to calculate the similarity levels between the
poplar genotypes and to generate a dendrogram. Polymorphism Information
Content (PIC value) of the AFLP markers was also calculated. Statistical analysis
of the AFLP data was carried out with the help of NTSYS-pc 2.0 (Rohlf, 1998) and
Microsoft Excel software packages. The data matrix was used to calculate genetic
similarity using three different coefficients namely- Nei and Li (1979), Sokal and
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
Michener (1958) and Jaccard (1908). The resulting distance matrices were
subjected to four clustering methods: UPGMA (unweighted pair group method
analysis; Sokal and Michener 1958), WPGMA (weighted pair group method
analysis; Sneath and Sokal 1973), complete linkage (Lance and Williams 1967),
and single linkage (Lance and Williams 1967). Two different data sets were used
for the analyses in this study: (1) All the 46 accessions, and (2) P. deltoides
accessions only (i.e. 43 P. deltoides without P. ciliata, P. maximowiczii and
P. euphratica). The cophenetic correlation coefficient was calculated to test the
goodness of fit between the similarity matrix and the cophenetic matrix (Sneath
and Sokal 1973). Correspondence between pairs of matrices was tested with the
Mantel Z statistic (Mantel 1967). Bootstrap analysis was also carried out.
Morphological data was available for some of the clones which was compared
with the genetic inter-relationhips revealed by AFLP.
4.2 Results
Ten selective AFLP primer combinations were employed for assessment of
genetic diversity in the 43 clones of P. deltoides along with three other species
namely, P. ciliata, P. maximowiczii and P. euphratica as controls (Table 4.1).
4.2.1 AFLP fingerprint profiles
A representative AFLP fingerprint obtained by primer combination E-AGC/M-
CTC is shown in Figure 4.1. This primer combination revealed 72 amplification
products of which 54 were polymorphic, showing 75% polymorphism. On
excluding the outliers, the P. deltoides clones accounted for 58 of the 72 bands
amplified by this primer combination. Thus, within P. deltoides there was only
43.1% polymorphism with 25 bands being polymorphic out of the 58 scored. As
can be seen in the fingerprint, the outliers (lanes = r, s, and t) are clearly
discernible from the rest of the P. deltoides clones. Several monomorphic and
polymorphic bands were generated by this primer combination (Figure 4.1). The
detected polymorphic bands were both frequent as well as rare in occurrence.
Amplified fragments which were absent in the outliers but monomorphically
present in the P. deltoides clones were seen in this fingerprint, such as the band
marked as ‘1’ in the figure (Figure 4.1). Polymorphic bands which were frequently
present in many genotypes were detected, such as those labelled as ‘2’ and ‘5’. The
band marked as ‘6’ denotes the polymorphic band which is infrequent in nature,
being present in only few of the genotypes. P. euphratica (lane= t) showed a very
distinct fingerprint pattern, which was very different from all the remaining
clones. In fact, there were many bands which were absent in P. euphratica but
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
monomorphic for all the remaining genotypes (e.g. band labelled ‘3’) and vice-a
versa. Two monomorphic bands are marked in Figure 4.1, labelled as ‘4’ and ‘7’.
Interestingly, an accession specific band was also identified by this primer
combination for the FRG clone A-26/75 (code = ‘j’) which is marked as an
arrowhead in the gel (Figure 4.1). This band can be converted to a SCAR marker,
which can be utilized for identification of this particular clone, if required.
Figure 4.2 represents a part of the AFLP fingerprint generated by the primer
combination E-ACC/M-CAG. This primer combination produced 55
amplification products showing 92.7% polymorphism. However, when the
outliers were excluded, 78.6% polymorhism was revealed among the P. deltoides
clones, which is highest percent polymorphism detected within P. deltoides.
Several polymorphic bands were revealed bby this primer combination such as
bands labelled as ‘1’ and ‘2’ in the gel (Figure 4.2), which are frequently present
among the analysed clones. This primer combination also detected bands that
were polymorphic but infrequent in nature, such as the band labelled as ‘3’ in the
Figure 4.2). The outlier, P. euphratica could be very distinctly differentiated with
the detection of P. euphratica specific markers such as the band labelled ‘4’
(Figure 4.2) which is present only in P. euphratica. There were bands that were
monomorphically present in all the clones except P. euphratica also, such as the
band labelled as ‘5’ in the photograph (Figure 4.2). This primer combination
revealed only four monomorphic bands of which one is labelled as ‘6’ in the
photograph (Figure 4.2). The monomorphic bands can be called as Populus
specific bands as they are present in four species of Populus representing three
different sections of the genus.
Figure 4.3 represents a part of the AFLP fingerprint generated by primer
combination E-AGG/M-CAA. Highest number of amplification products (75) was
obtained using this primer combination. This primer combination revealed
89.3% polymorphism, which reduced to 65.6% on exclusion of the outliers. A
total of 61 bands were amplified by the P. deltoides clones, which is the highest
number revealed by the use of any of the ten primer combinations. The outliers
were clearly discernible in this fingerprint as well. Bands labelled as ‘1’ is the
markers which is monomorphically present in all the clones except P. euphratica
(Figure 4.3). This marker can be used for distinguishing P. euphratica and can be
designated as species specific diagnostic marker. Similarly, a P. ciliata specific
null marker was also amplified which is labelled as ‘2’ and is monomorphically
present in all the clones except P. ciliata (Figure 4.3). Several polymorphic bands
(such as those labelled as ‘3’ and ‘4’) were identified in the gel profile (Figure 4.3).
The monomorphic markers were also detected by this primer combination, such
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
as the band labelled as ‘5’ in the fingerprint (Figure 4. 3). An interesting marker
was observed with respect to the clone ‘UHF-55-2’ (lane = R) in the fingerprint.
The arrowhead marks the absence of amplification in the clone ‘UHF-55-2’ in an
otherwise monomorphic band (Figure 3) showing this clone to be distinct.
Similar fingerprint profiles were obtained using ten different primer
combinations and 0/1 matrix was obtained by manual scoring for each of the
primer combinations. Only the distinct bands were scored and in case of any
ambiguity, the marker was not included for further analysis
4.2.2 Statistical Analysis of data
The AFLP bands of all the 10 primer combinations were scored for polymorphism
across the 46 genotypes of Populus. A total of 601 amplified fragments were
identified. A very high number of polymorphic bands were obtained with all the
primer combinations. Table 4.2 lists the summary of results in terms of number
of bands obtained per primer combination and the percent polymorphism. The
analysis indicated 518 polymorphic bands out of a total of 601 bands, which
revealed 86.3% polymorphism. The high level of polymorphism detected was due
to the fact that different species were used as outliers in the study (Table 4.2).
The highest number of bands (75) were generated by E-AGG/M-CAA primer
combination, whereas least number of bands (46) were generated by E-ACC/M-
CTG primer combination. On an average, 60 bands was obtained per assay of
which 52 were polymorphic (Table 4.2). The banding pattern clearly
distinguishes the P. deltoides clones from the three other-outlier Populus species.
Comparative analysis of the polymorphism among P. deltoides and
between Populus species, which were used as outliers, was carried out. Table 4.2
shows the polymorphism obtained within P. deltoides and that obtained from P.
deltoides, P. maximowiczii, P. ciliata and P. euphratica together. In the dataset
comprising only of P. deltoides clones, a total of 444 bands were scored of which
241 were polymorphic. The primer combination E-AGG/M-CAA produced the
highest number of bands (61) and revealed 65.6% polymorphism. The primer
combination E-ACC/M-CAG showed highest percent polymorphism (78.6%)
within P. deltoides. On an average 44 bands were amplified per primer
combination and 54.1% polymorphism was detected within the P. deltoides
clones (Table 4.2).
4.2.2.1 Calculation of Polymorphic Information Content
The polymorphic information content (PIC) for each of the AFLP marker was
calculated and the average was found to be 0.14 and 0.20 for the complete
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
dataset and the P. deltoides dataset, respectively. The PIC value can fall between
the range of a minimum of ‘zero’ to a maximum of ‘0.5’ with a higher value
indicating more informative polymorphism. In our study, most of the AFLP
markers showed a low PIC value of 0.01 to 0.10, followed by those showing the
PIC value of 0.11 to 0.20 and 0.41 to 0.50 (Figure 4.4A and 4.4B). The number of
AFLP markers showing different PIC values are depicted as graphs in Figure 4.4A
and 4.4B for the complete dataset and P. deltoides dataset, respectively. Least
number of AFLP markers showed the PIC value of 0.31 to 0.40 (Figure 4A and
4B). These values show that maximum number of markers had low PIC value and
only a few markers were actually more informative.
4.2.2.2 Construction of Similarity Matrix
Initially three similarity matrices were calculated on two data sets using Nei and
Li’s, Jaccard’s and Sokal and Michener’s coefficients, respectively. The two data
sets differed in over all levels of similarly value, but there was close agreement
between the relative values of the three coefficients. The three similarity matrices
were subjected to four different clustering methods (UPGMA, WPGMA, complete
linkage and single linkage) and cophenetic correlations were calculated which are
provided in Table 4.4. The cophenetic correlations were found to be very strong
(more than 0.98) for all the matrices generated by the complete dataset (Table
4.4). However, the cophenetic correlation values were low for the P.deltoides
dataset (less than 0.79) indicating the usefulness of outliers in the study (Table
4.4). The Mantel Z statistic was calculated for comparing the different similarity
coefficients in both the datasets. All the coefficients were found to be well
correlated with each other (Table 4.5). Jaccard’s coefficient showed highest
correlation with Sokal and Michener’s coefficient and Nei and Li’s coefficient for
the complete and P.deltoides datasets, respectively (Table 4.5). In conclusion,
Jaccard’s similarity coefficient and UPGMA cluster analyses were found to give
high cophenetic correlation for the analysis irrespective of the datasets being
used. Hence, in the final analysis only the UPGMA clustering method and
Jaccard’s similarity coefficient were considered.
Jaccard’s similarity value (GSJ) for the complete data set (1035 pairs) was
found to range from 0.301 to 0.977, with an average of 0.814 (Table 4.3). The
highest similarity was found between the accessions ‘200/86’ and 200/85’,
whereas the lowest similarity was between ‘65/27’ and P.euphratica. In the
P. deltoides data set, the GSJ ranged from 0.777 to 0.977, with an average of
0.933. The highest similarity was found between the accessions ‘200/86’ and
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
200/85’, whereas the lowest similarity was between ‘45/51’ and ‘UHF-55-2’. The
outliers shared very less similarity with the P. deltoides clones with and average
of 0.426 GSJ.
4.2.2.3 Cluster analysis
A dendrogram was constructed with UPGMA cluster analysis based on Jaccards
similarity coefficient, which is shown in Figure 4.5. In the dendrogram the
outgroups P. maximowiczii, P. ciliata and P.euphratica separated out very
clearly from all the P. deltoides genotypes. The dendrogram shows that
P. euphratica clustered out first at 0.32 similarity value from the remaining
genotypes, followed by P. maximowiczii and P. ciliata at 0.51 similarity value
(Figure 4.5). The different groups of P. deltoides representing different sources of
origin (Table 4.1) intermingled with each other and formed a separate cluster
(Figure 4.5). The ‘UHF-55-2’ accession, one of the UHF selection from the USA
seemed to be very distinct from rest of the P. deltoides clones as it separated out
very distinctly at 0.83 similarity value (Figure 4.5). The LalKuan clone ‘L-29/82’ ,
which is one of the seven clones identified as better performing among those
raised from G-48 X G-3 cross in 1982, also separated out distinctly (Figure 4.5).
Four more clones separated out one after the other from the remaining clones
namely, UHF-65-2, 3295-NL, 45/51 and L-181/84 (Figure 4.5). Within the
remaining P. deltoides clones, the three clones of the selections of 86/85
(‘126/86’, ‘200/86’, and ‘200/85’) grouped together in the dendrogram (Figure
4.5). Four clones of the LalKuan series (‘L-34/82, ‘L-13’, ‘L-52/82’ and ‘L-75/84’)
were close to the clone ‘UHF-51’ and one (‘L-165/84’) clustered with three clones
from the USA (S7C8, S7C20 and IC) in the dendrogram (Figure 4.5). The three
clones ‘PD-345/183’, ‘C-181’ and ‘A-50 (89)’which were of unknown origin
clustered together along with the clone ‘65/27’ which was from Australian. All the
remaining clones intermingled with each other in the dendrogram and no
definite groupings were observed, with only some UHF selections clustering
together namely ‘UHF-47A, ‘UHF-63’ and ‘UHF-99’ (Figure 4.5). A UPGMA
dendrogram was also constructed for the P. deltoides dataset, which revealed
almost similar clustering pattern.
4.2.2.4 Principal Component Analysis
Principal Component Analysis (PCA) was carried out by calculating the
correlation matrix and eigen values of the complete dataset and the P.deltoides
dataset. The two-dimensional (2D) and three dimensional (3D) scatter plots
obtained by PCA are shown in Figure 4.6 and 4.7 respectively. The Figures 4.6A
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
and 4.6B show the 2D and 3D plots of the complete dataset, and the Figures 4.7A
and 4.7B show the 2D and 3D plots of the P. deltoides dataset, respectively. PCA
plots of the complete dataset distinguished the outliers and the P. deltoides
genotypes which grouped very close (Figure 4.6A and 4.6B). The first three axes
in the PCA plot accounted for 72.84% of variation. When a PCA plot was
constructed using the P. deltoides dataset (Figure 4.7A and 4.7B), the first three
axes explained only 26.64% of variation. Mantel test revealed that the
dendrogram shown in Figure 4.5, was a near true representation of the similarity
matrix generated by Jaccard’s coefficient, indicated by high cophenetic
correlation value, r = 0.9904, P < 0.01. The cophenetic correlation value was
found to show a poor fit (0.7991) for the dataset without the outliers. Bootstrap
values were 100% for the outliers separating out from the P. deltoides clones, but
low within the groupings of P. deltoides (Figure 4.8).
The morphological data was found to somewhat correlate with the genetic
distinctness found in the present study and has been discussed in detail in the
next section of discussions. The results indicate low level of genetic variation
within the P. deltoides clones grown in India.
4.3 Discussions
The present study had three objectives (i) To assess the genetic variation within
P. deltoides clones grown in India; (ii) to test the correlation between clone
identification based on source of introduction, and (iii) to identify potential
resources for future germplasm management. The usual practice for clone
identification is the use of UPOV guidelines based on morphology, but the
interpretations are difficult as the edaphic conditions have overriding influence
on the genotype. The use of molecular systematics for the evaluation of genetic
diversity thus becomes essential for breeding programs. Several workers have
shown that the classification by AFLP markers is more reliable than
morphological classification, where characters are influenced by environmental
effects (Cervera et al. 1998). Therefore, AFLP was utilized in the present study for
genetic diversity evaluation of P. deltoides clones. This required informative and
reproducible AFLP banding pattern where primer selection is very important
both in the pre-amplification and in the selective amplification steps. High
multiplex ratio is desirable for molecular marker assays. Multiplex ratio is the
number of loci (or bands) simultaneously analyzed per assay or primer
combination (Powell et al. 1996). The length of the 3’ extension of the primer
modulates the multiplex ratio in AFLPs (Zabeau and Vos 1993). For genomes
larger than 0.06pg/2C and up to 2.00pg/2C, a pre-amplification of EcoRI+1 &
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
MseI+1 followed by a selective amplification with EcoRI+3 & MseI+3 is required
(Cervera et al. 2000). These primer selections have already been screened in
poplars (Cervera et al. 2000, Chauhan et al. 2003), hence similar strategy was
employed in the present study.
4.3.1 Statistical Analysis
Several AFLP primer combinations were tested on few P. deltoides samples and
ten most informative ones were taken for screening all 46 samples. Three other
poplar species namely, P. ciliata, P. maximowiczii and P. euphratica were
included in the study mainly to be used as outliers with the 43 P. deltoides clones
(Table 4.1). The incorporation of outliers was to obtain a precise clustering
pattern in the dendrogram. Further, use of outliers helped in accurately
estimating similarity within P. deltoides. It has been found in soybean (Powell et
al. 1996) and buffalograss (Peakall et al. 1995) that when two divergent genotypes
are used, the marker density is sufficient to adequately sample genetic diversity
between more distant individuals. The use of these outliers was helpful in our
analysis.
The inter-specific variation in poplars has previously been reported to be
high with 91 and 94 percent polymorphic AFLP fragments in P. deltoides vs
P. trichocarpa and P. deltoides vs P. nigra respectively (Cervera et al. 2000). The
main objective of the present study was to assess the intra-specific genetic
variation and not the interspecific diversity, hence only a single representative of
the three outlier species were included in the experiment.
The intra-specific variability in 15 clones of P. deltoides has been
investigated by using a single AFLP primer combination namely E-AGC & M-TGT
by Cervera et al. (2000). They detected 61% of the AFLP fragments (out of 54) to
be polymorphic. The slightly higher report of polymorphism in P. deltoides by
Cervera et al. (2000) could have resulted from either the use of different
accessions (Their 15 clones were different from the 43 clones analyzed in the
present study) or the use of different primer for pre-amplification (Mse+T
compared to Mse+C in the present study). However, out of the ten primer
combinations used, the percent polymorphism detected was below 50% in four
primer combinations, in two primer combinations between 50% to 60%, and in
the remaining four above 60% (Table 4.2). Thus, in four primer combinations
similar levels of polymorphism was detected as earlier reported by Cervera et al.
(2000) in P. deltoides clones.
The PIC value of each marker represents the probability of finding this
marker in two different states (present/absent) in two genotypes drawn at
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
random from the population. AFLP markers being dominant in nature and
having only two states namely, present and absent, the highest PIC value cannot
exceed 0.5. The PIC value ranges from zero for monomorphic markers to 0.5 for
markers that are present in 50% of the genotypes and absent in the other 50%.
The lower PIC value in the complete datset (0.14 as compared to 0.20 in
P. deltoides dataset) was mainly due to the fact that only three genotypes
(outliers) were accounting for a total of 157 bands. If more clones of the three
outlier species had been incorporated, we predict that the PIC would be more or
equal to that of the P. deltoides dataset. The average PIC value of AFLP markers
in P. deltoides is comparable to those obtained in different plant species such as
soybean (0.32; Powell et al. 1996 ) and ryegrass (0.17-0.23; Roldan-Ruiz et al.
2000). The moderate value of the average PIC value (0.20) of AFLP markers
indicate that these are adequate for assessing genetic variation in P. deltoides.
High genetic similarity values were revealed on examination of the
Jaccard’s similarity matrix shown in Figure 3, with an average of 0.814 and 0.933
for the complete dataset and the P. deltoides dataset respectively. The use of the
three outliers can be assumed in contributing towards this difference. In another
study, the GSJ values based on AFLP markers was found to range from 0.522 to
0.971 between P. ciliata, P. maximowiczii and their interspecific hybrids, with
0.5222 to 0.586 GSJ between P. ciliata and P. maximowiczii species (Chauhan et
al. 2003). With a much less GSJ value between P. deltoides and the outliers
(0.426 GSJ), greater genetic distance is revealed between them, indicating them
to be ideal for inter-specific hybridization.
In the UPGMA dendrogram (Figure 4.5) the outliers P. euphratica
separated out very distinctly followed by the two species cluster of P. ciliata and
P. maximowiczii. The separating out of P. euphratica is supported taxonomically
as it is the monotypic species of the Section Turanga of the genus Populus, and is
reported to be very distant from rest of the poplar species (Chardenon and
Semizoglu 1965). The grouping together of P. maximowiczii and P. ciliata is also
supported taxonomically as they belong to the same section namely Tacamahaca
(Eckenwalder 1996). The different groups of P. deltoides representing different
sources of introduction (Table 4.1) intermingled with each other and formed a
separate cluster (Figure 4.5).
4.3.2 Comparison with morphological characters
Morphological data for the clones of UHF selections from the USA (clones
N to W; Table 4.1) and the 85/86 selections (clones A, B and C; Table 4.1) was
available (Khurana and Mohanty 2000) and was compared with the patterns
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
observed in the dendrogram. The clones of the 85/86 selections grouped together
whereas those of the UHF selections intermingled in the dendrogram showing no
definite clustering pattern (Figure 4.5). The general shape of the leaf blade was
found to be unlobed in all the clones and the shape of the base of the leaf blade
was weakly-cordate with the exception of two clones namely ‘200/85’ and ‘UHF-
PD-90’ where it was cordate (Khurana and Mohanty 2000).
The clone ‘UHF-55-2’ was found to be genetically (Figure 4.5) as well as
morphologically very distinct from all the clones of the UHF selection and 85/86
selections. The color of the upper side of the leaf when flushing was brown-green
for the clone ‘UHF-55-2’, whereas for rest of the clones it was green (Khurana and
Mohanty 2000). Further, all the clones were found to possess two glands at the
base of the leaf blades whereas ‘UHF-55-2’ had more than two glands. Glands at
the base of leaf have been used for identification of one year poplar clones in the
nursery (Broekhulzen 1964). This character thus makes ‘UHF-55-2’
morphologically identifiable as distinct, confirming the genetic variability
observed in the present study.
Khurana and Mohanty (2000) reported that all the clones had narrow
accuminate tip of the leaf blade with the exception of ‘UHF-55-2’ and ‘126/86’,
which had broad accuminate tips. The clone ‘126/86’ was also different in
possessing important undulations in the leaf blade-edges whereas rest of the
clones have minor undulations (Khurana and Mohanty 2000). Broekhulzen
(1964) used leaf blade undulation for characterization of one-year-old plants of
poplars, making it an important character for morphological identification. It is
clear from the dendrogram that the clone ‘126/86’ is genetically distinct not only
from the UHF selections but also from the other 85/86 selections (Figure 4.5).
Another UHF clone to separate out distinctly in the dendrogram was ‘UHF-65-2’
(Figure 4.5). This clone had a distinguishing characteristic of presence of slight
pubescence on the lower surface of the leaf blade whereas in the rest of the clones
pubescence was found in the whole surface of the leaf (Khurana and Mohanty
2000). Further, pilosity, which is the morphological trait of the petiole, was
restricted to the upper part of petiole in all the clones except ‘UHF-65-2’ where
no pilosity was observed (Khurana and Mohanty 2000).
It was thus observed that AFLP can distinguish the poplar clones which
are morphologically distinct. A correlation between the morphological character
of height-growth and AFLP markers has also been reported in the case of
P. ciliata X P. maximowiczii hybrids (Chauhan et al. 2003). Hence, this study
shows that AFLP markers can be used for screening and selection of distinct
clones in various poplar breeding programs.
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
4.3.3 Genetic variation in P.deltoides
The results indicate that there is low genetic diversity within the
P. deltoides clones grown in India (0.777-0.977 similarity value), considering they
show morphological variation and have been introduced/ developed from
different sources. There could be two reasons of this narrow genetic base: (1)
there is low genetic variation within P. deltoides itself or, (2) it is also possible
that some labeling/identification error of the exotic P. deltoides clones would
have occurred during the time of their introduction or propagation and
duplicates/hybrids have been labelled as P. deltoides.
Artificial selection and domestication result in reduced genetic diversity.
P. deltoides is known to reproduce primarily by seeds in nature, and because of
its dioecious nature (Hu et al. 1985), outcrossing is obligatory. The inherent
genetic diversity is thus expected to be high. But there has been a long history of
domestication and breeding in Populus. A large pool of commercially available
clones/cultivars has resulted from a strong emphasis on vegetative propagation
(Dickmann and Isebrands 2001). As most of the poplar species are cultivated
clonally through vegetative propagation, selection of a narrow genetic base
becomes inevitable. There could be inherent low genetic diversity in the
P. deltoides clones grown in India as mostly the selections/collections have been
from the southern States of the USA where the climatic conditions are similar to
India. Few clones accidently got selected, which suited to the Indian conditions,
and they happen to mostly represent the Texas, Louisiana and Mississippi States
of the USA. Exploitation of the high interspecific variation, for poplar breeding
programmes, thus becomes essential to increase the genetic base of P. deltoides.
Mislabelling of clones is likely to be another important reason for the
detection of low genetic diversity within the P. deltoides clones. One of the major
concerns in poplar germplasm maintenance is the incorporation of duplicates
and hybrids in the gene banks. In case of P. nigra gene bank collections in
Europe, it has been reported that 26% of the accessions were duplicates (Storme
et al. 2003). In our study, we did not find any duplicates, which indicates that
each clone is genetically distinct. Further, the clones which were studied, are
maintained at three places by the Dr. YS Parmar University of Horticulture and
forestry, Nauni and at two places by the Forest Research Institute, Dehradun,
hence mislabelling cannot occur easily. However, the possibility of presence of
hybrids labelled as P. deltoides cannot be ruled out. The Australian clone ‘65/27’,
which clustered with three clones of unknown origin in the dendrogram namely
‘PD-345/183’, ‘C-181’ and ‘A-50 (89)’, is of interest in this context (Figure 4.5).
The clone ‘65/27’ has been suspected and reported by some Indian foresters as
P. deltoides genetic diversity
TERI School-Ph.D Thesis, 2004
not being pure P. deltoides but hybrid with P. nigra namely P. X eumericana
(Mehta et al. 1995). The grouping of ‘65/27’ with the three clones from unknown
source of introduction, raises a question to the identity of the three clones as pure
P. deltoides. There is evidence that some clonal selections of poplar interspecific
hybrids may be misidentified as P.deltoides due to their similarity in
morphological traits (Rajora and Zsuffa, 1991).
Another interesting possibility is that the clones would have been
misidentified before introduction into the country. The clones are identified on
the basis of morphological characters as provided in the UPOV guidelines. Earlier
Rajora and Zsuffa (1991) had suggested that morphological method of
P. deltoides identification is not completely reliable. They had found 16% of the
169 P. deltoides lines (selections made in Canada and U.S.A.) screened by
allozymes, were not pure P. deltoides. These were identified as the interspecific
hybrids P. X canadensis and P. X jackii. These hybrids vary in morphology and
are easily mistaken for P. deltoides. Therefore, one cannot be sure of the purity of
the introduced Populus clones, if genetic identification is not provided along with
the morphological standards. The clones ‘UHF-55-2’, ‘L-29/82’, ‘UHF-65-2’, ‘L-
181/84’, ‘45/51’ and ‘3295-NL’ were found very distinct from rest of the
P. deltoides clones, and need to be tested for true identity. It is possible that these
clones, and particularly ‘65/27’ along with ‘PD-345/183’, ‘C-181’ and ‘A-50 (89)’,
are not ascertained their true parentage and further investigation is necessary to
know if they are pure P. deltoides.
Table 4.1 List of Populus clones analyzed for genetic diversity study
S.No Species Clone Source Origin
1 A P.deltoides 126/86 Selection of 85/86 India+ 2 B P.deltoides 200/86 Selection of 85/86 India+ 3 C P.deltoides 200/85 Selection of 85/86 India+ 4 D P.deltoides L-52/82 LalKuan series India+ 5 E P.deltoides L-34/82 LalKuan series India+ 6 F P.deltoides L-13 LalKuan series India+ 7 G P.deltoides L-75/84 LalKuan series India+ 8 H P.deltoides L-181/84 LalKuan series India+ 9 I P.deltoides L-165/84 LalKuan series India+ 10 J P.deltoides L-29/82 LalKuan series India+
11 K P.deltoides G-3 Australia USA
12 L P.deltoides G-48 Australia USA
13 M P.deltoides D-121 USA USA
14 N P.deltoides UHF-Pd-90 UHF selection of USA USA
15 O P.deltoides UHF-14—2 UHF selection of USA USA
16 P P.deltoides UHF-47-A UHF selection of USA USA
17 Q P.deltoides UHF-51 UHF selection of USA USA
18 R P.deltoides UHF-55-2 UHF selection of USA USA
19 S P.deltoides UHF-60-3 UHF selection of USA USA
20 T P.deltoides UHF-65-2 UHF selection of USA USA
21 U P.deltoides UHF-63 UHF selection of USA USA
22 V P.deltoides UHF-100-9 UHF selection of USA USA
23 W P.deltoides UHF-99 UHF selection of USA USA
24 X P.deltoides S7C48 Stoneville, USA USA
25 Y P.deltoides S7C8 Stoneville, USA USA
26 Z P.deltoides S7C20 Stoneville, USA USA
27 a P.deltoides 26152–NL Netherlands USA
28 b P.deltoides PD-2498–NL Netherlands USA
29 c P.deltoides PD-365–NL Netherlands USA
30 d P.deltoides 3295–NL Netherlands USA
31 e P.deltoides 3298–NL Netherlands USA
32 f P.deltoides 2652–NL Netherlands USA
33 g P.deltoides PD-2094 Unknown Unknown
34 H P.deltoides 9/54-9-FRG FRG* USA
35 i P.deltoides 194(38)–FRG FRG* USA
36 j P.deltoides A-26/75–FRG FRG* USA
37 k P.deltoides PD-345/183 Unknown Unknown
38 l P.deltoides G-113324 USA USA
39 m P.deltoides IC USA UK
40 n P.deltoides 45/51 Unknown Unknown
41 o P.deltoides A-50(89) Unknown Unknown
42 p P.deltoides C-181 Unknown Unknown
43 q P.deltoides 65/27 Australia Australia
44 r P.ciliata Pc-1 India India
45 s P.maximowiczii Pm-6 Italy Japan
46 t P.euphratica Pe-70 India India +Clone developed in India by breeding of exotic P.deltoides clones G3 and G48 *Federal Republic of Germany
Table 4.2 Total number of AFLP bands obtained by using different primer combinations for the two data-sets (with and without controls)
Primer Combi-nation
P.deltoides and P.ciliata+
P.euphratica+ P.maximowiczii Populus deltoides
Total no. of Bands
Mono-
morphic bands
Poly-
morphic bands
% Poly-
morphism
Total no.of
Bands
Mono
Morphic bands
Poly-
Morphic bands
% Poly-
morphism
E-ACC,
M-CAG
55 4 51 92.7 42 9 33 78.6
E-ACC,
M-CTG
46 10 36 78.3 37 18 19 51.4
E-ACC,
M-CTC
56 4 52 92.9 46 22 24 52.2
E-AGC,
M-CAT
60 5 55 91.7 48 18 30 62.5
E-AGC,
M-CTC
72 18 54 75.0 58 33 25 43.1
E-AGG,
M-CAA
75 8 67 89.3 61 24 40 65.6
E-ACT,
M-CAC
61 7 54 88.5 37 14 23 62.5
E-ACT,
M-CTG
55 8 47 85.5 37 20 17 45.9
E-AAG,
M-CAC
57 9 48 84.2 31 17 14 45.2
E-AGG,
M-CTT
64 10 54 84.4 47 31 16 34.0
Total 601 83 518 444 206 241
Average 60.1 8.3 51.8 86.3 44.4 20.6 24.1 54.1
Table 4.3 Cophenetic Correlation Values for comparison of different clustering methods and similarity coefficients
Clustering Method Similarity Coefficients
Sokal & Michener’s Jaccard's Nei & Li's
Complete dataset UPGMA 0.99401 0.99264 0.99525 WPGMA 0.99170 0.98926 0.99395 Single Linkage 0.99186 0.99151 0.99465 Complete Linkage 0.98811 0.98578 0.99147 P. deltoides dataset UPGMA 0.78295 0.79171 0.78621 WPGMA 0.77727 0.74304 0.77386 Single Linkage 0.75119 0.76862 0.76701 Complete Linkage 0.67417 0.67771 0.67615
Table 4.4 Mantel Z Statistic for comparison of different similarity coefficients
Nei & Li's Jaccard's Sokal & Michener’s
Nei & Li's * 0.99653 0.99818
Jaccard's 0.99973 * 0.99875
Sokal & Michener’s 0.99602 0.99638 *
Data above the diagonal is for the complete dataset and below diagonal is for the P.deltoides dataset
Table 4.5 Jaccard’s similarity matrix of the 43 Populus clones along with the three outgroup species.
1.0000000 0.8906250 1.0000000 0.8850932 0.9771429 1.0000000
0.8237082 0.8449848 0.8510638 1.0000000 0.8308157 0.8852459 0.8907104 0.9000000 1.0000000 0.8693009 0.8997290 0.9051491 0.8984615 0.9205479 1.0000000 0.8515152 0.9019074 0.9021739 0.8861538 0.9175824 0.9531680 1.0000000
0.8006042 0.8387097 0.8440860 0.8287462 0.8536585 0.8885870 0.8753388 1.0000000 0.8303571 0.8847185 0.8850267 0.8417910 0.8796791 0.8938992 0.9010695 0.8488064 1.0000000 0.8095238 0.8519637 0.8666667 0.8596491 0.9037267 0.8742515 0.8652695 0.8005952 0.8299120 1.0000000 0.8377483 0.8439306 0.8497110 0.8750000 0.9101796 0.9144543 0.9000000 0.8488372 0.8494318 0.8552632 1.0000000
0.8750000 0.8905775 0.8966565 0.8640483 0.9051988 0.9268293 0.9146341 0.8632219 0.9036145 0.8698630 0.8907285 1.0000000 0.8402367 0.9003021 0.9006024 0.8795181 0.9033233 0.9131737 0.9184290 0.8507463 0.9074627 0.8552189 0.8733766 0.9074627 1.0000000 0.8327645 0.8909091 0.8969697 0.8819444 0.9024390 0.9240122 0.9063444 0.8528529 0.8928571 0.8682432 0.8943894 0.8938356 0.8888889 1.0000000 0.8378378 0.8582888 0.8586667 0.8834356 0.8937330 0.9029650 0.9155313 0.8471850 0.8680739 0.8363095 0.8914956 0.8945783 0.9041916 0.9033233 1.0000000
0.8487973 0.8993902 0.8996960 0.8919861 0.9110429 0.9386503 0.9033233 0.8610272 0.8842730 0.8745763 0.9102990 0.9037801 0.8986486 0.9174312 0.9060606 1.0000000 0.7841945 0.8086253 0.8140162 0.8235294 0.8587258 0.8432432 0.8301887 0.7876344 0.8095238 0.8478261 0.8486647 0.8527607 0.8184524 0.8343373 0.8172043 0.8536585 1.0000000 0.8519737 0.8774834 0.8841060 0.8733333 0.9060403 0.9133333 0.9033333 0.8856209 0.8729642 0.8426966 0.8795620 0.8885246 0.9013158 0.9118774 0.8910891 0.9083969 0.8278146 1.0000000 0.8561644 0.8768769 0.8828829 0.8338983 0.8825301 0.8895522 0.8892216 0.8654971 0.8596491 0.8523490 0.8612903 0.8624161 0.8566667 0.9013605 0.8609467 0.8787879 0.8017751 0.8695652 1.0000000
0.7994012 0.8121693 0.8222812 0.8272727 0.8463612 0.8709677 0.8629032 0.8058511 0.8082902 0.8160237 0.8684211 0.8447761 0.8328446 0.8532934 0.8449198 0.8614458 0.7951482 0.8214286 0.8338279 1.0000000 0.8195719 0.8532609 0.8536585 0.8656250 0.8736264 0.8934426 0.8801090 0.8419619 0.8582888 0.8288288 0.8613569 0.8378378 0.8588589 0.8895706 0.8719346 0.8923077 0.8065395 0.8662207 0.8670695 0.8297297 1.0000000 0.8401254 0.8694444 0.8674033 0.8666667 0.9011299 0.9052925 0.9050279 0.8472222 0.8870523 0.8468468 0.8821752 0.8871473 0.8913043 0.8850932 0.8805556 0.9056604 0.8291317 0.8801370 0.8680982 0.8555556 0.8866856 1.0000000 0.8695652 0.8695652 0.8699187 0.8641975 0.8953168 0.9046322 0.8964578 0.8583106 0.8743316 0.8489426 0.8875740 0.8929664 0.8798799 0.9021407 0.9090909 0.9107692 0.8228883 0.8741722 0.8939394 0.8459459 0.9154930 0.9116809 1.0000000
0.8428094 0.8533333 0.8571429 0.8673469 0.8817568 0.9060403 0.8989899 0.8227425 0.8552632 0.8401361 0.8805970 0.8770764 0.8936877 0.8740458 0.8733333 0.8961538 0.7906977 0.8750000 0.8603774 0.8720539 0.8771331 0.9075342 0.8907850 1.0000000 0.8417910 0.8927614 0.8981233 0.8644578 0.9029650 0.9069149 0.9193548 0.8766756 0.9327957 0.8441176 0.8793103 0.9096386 0.9307229 0.9044776 0.8859416 0.8958333 0.8320000 0.8827362 0.8713450 0.8398950 0.8713137 0.9217877 0.8975741 0.8929766 1.0000000 0.8268657 0.8643617 0.8647215 0.8662614 0.8643617 0.8835979 0.8856383 0.8533333 0.9090909 0.8294118 0.8518519 0.8888889 0.8985075 0.8813056 0.8627968 0.8783383 0.8138298 0.8603896 0.8405797 0.8315789 0.8579088 0.8763736 0.8790323 0.8700000 0.9171123 1.0000000 0.8429003 0.8528529 0.8507463 0.8658537 0.8757576 0.9115854 0.8966565 0.8313253 0.8772455 0.8338870 0.8671096 0.8772455 0.9039039 0.8831615 0.8851964 0.8737201 0.8042169 0.8870432 0.8639456 0.8438438 0.8650307 0.8940810 0.8834356 0.8907285 0.8975904 0.8795181 1.0000000
0.8318318 0.8510638 0.8563830 0.8888889 0.9120879 0.9211957 0.9078591 0.8648649 0.8657895 0.8614458 0.9427711 0.8885542 0.8813056 0.9090909 0.8997290 0.9176829 0.8297297 0.8881579 0.8727811 0.8475936 0.8695652 0.8753463 0.8858696 0.8855219 0.8885942 0.8753316 0.8791541 1.0000000 0.8658537 0.8967391 0.9021739 0.9068323 0.9228650 0.9371585 0.9237057 0.8804348 0.8759894 0.8674699 0.9058824 0.8945783 0.9156627 0.9151515 0.9051491 0.9296636 0.8351351 0.9100000 0.8978979 0.8729730 0.8956044 0.9075630 0.9068493 0.9050847 0.9193548 0.8906667 0.8939394 0.9234973 1.0000000 0.8328358 0.8350785 0.8403141 0.8224852 0.8350785 0.8590078 0.8511749 0.8051948 0.8402062 0.8110465 0.8376068 0.8613569 0.8439306 0.8454810 0.8290155 0.8479532 0.8042328 0.8349206 0.8280802 0.8219895 0.8284960 0.8482385 0.8541114 0.8552632 0.8720627 0.8590078 0.8575668 0.8507853 0.8560209 1.0000000 0.8355263 0.8505747 0.8505747 0.8509934 0.8637681 0.9067055 0.8815029 0.8352601 0.8821839 0.8225806 0.9035370 0.8631922 0.8705502 0.8827362 0.8703170 0.8918033 0.7908309 0.8800000 0.8498403 0.8357349 0.8600583 0.8761329 0.8882353 0.8703704 0.8879310 0.8760807 0.8833333 0.8921283 0.8811594 0.8681948 1.0000000
0.8214286 0.8425926 0.8430769 0.8469751 0.8637771 0.8885449 0.8878505 0.8240741 0.8742331 0.8219178 0.8737201 0.8591549 0.9007092 0.8904594 0.8753894 0.8701754 0.8000000 0.8577075 0.8631579 0.8307692 0.8504673 0.8935484 0.8718750 0.8943089 0.9009288 0.8978328 0.9481481 0.8788820 0.8937500 0.8541033 0.8809524 1.0000000 0.8424242 0.8484043 0.8537234 0.8654434 0.8632708 0.9029650 0.8796791 0.8422460 0.8730159 0.8338279 0.8859649 0.8545994 0.8698225 0.8975904 0.8766756 0.9003021 0.7978723 0.8907285 0.8613569 0.8400000 0.8668478 0.8781163 0.8882834 0.8821549 0.8859416 0.8826667 0.8844985 0.8997290 0.8948787 0.8627968 0.9575758 0.8909657 1.0000000 0.8238095 0.8960000 0.9076305 0.8829268 0.8947368 0.9193548 0.9116466 0.8473896 0.9090909 0.8691589 0.8783784 0.8851675 0.9326923 0.8964143 0.8920000 0.9072581 0.8320000 0.9252874 0.8785047 0.8452381 0.8785425 0.8991597 0.8906883 0.8857143 0.9280000 0.9314516 0.9029126 0.9032258 0.9629630 0.8390805 0.8868778 0.9086294 0.8995984 1.0000000 0.8424242 0.8408488 0.8461538 0.8597561 0.8655914 0.9054054 0.8870968 0.8395722 0.8654354 0.8147059 0.8918129 0.8601190 0.8643068 0.8802395 0.8840970 0.9000000 0.7904509 0.8907285 0.8584071 0.8422460 0.8540541 0.8701657 0.8855586 0.8758389 0.8684211 0.8700265 0.8902439 0.8970190 0.8972973 0.8552632 0.9425982 0.8937500 0.9525140 0.8840000 1.0000000
0.8106509 0.8541114 0.8594164 0.8549849 0.8891892 0.8933333 0.8753316 0.8529412 0.8590078 0.8422619 0.9008746 0.8609467 0.8761062 0.8753709 0.8773333 0.9006024 0.8133333 0.8697068 0.8483965 0.8215223 0.8575269 0.8732782 0.8636364 0.8666667 0.8865435 0.8783069 0.8516320 0.9157609 0.9211957 0.8251928 0.8678161 0.8562691 0.8873995 0.9271255 0.8746667 1.0000000 0.8102410 0.8297872 0.8351064 0.8272727 0.8445040 0.8689840 0.8709677 0.8090186 0.8302872 0.8053097 0.8550725 0.8284024 0.8601190 0.8486647 0.8579088 0.8456973 0.7983871 0.8333333 0.8482143 0.8164894 0.8328841 0.8434066 0.8540541 0.8372093 0.8624339 0.8590426 0.8549849 0.8706199 0.8760108 0.8346457 0.8575581 0.8875000 0.8679245 0.8764940 0.8702703 0.8439153 1.0000000 0.8250825 0.8625731 0.8629738 0.8682432 0.8846154 0.8947368 0.9026549 0.8676471 0.8895349 0.8306189 0.8908555 0.8903654 0.8668831 0.8848684 0.8775510 0.9069767 0.8187135 0.8722628 0.8520900 0.8439306 0.8805970 0.8648649 0.8902077 0.8694030 0.8982558 0.8976608 0.8633333 0.9109792 0.9085546 0.8400000 0.8730159 0.8664384 0.8720930 0.9009009 0.8833819 0.8979592 0.8414986 1.0000000 0.8459215 0.8663102 0.8617021 0.8689024 0.8563830 0.8856383 0.8776596 0.8552279 0.9164420 0.8230088 0.8490028 0.8858859 0.8898810 0.8672566 0.8451444 0.8809524 0.8106667 0.8571429 0.8425656 0.8141361 0.8400000 0.8681319 0.8659517 0.8448845 0.9090909 0.9214092 0.8597015 0.8526316 0.8877005 0.8463542 0.8649425 0.8676923 0.8696809 0.9120000 0.8620690 0.8507853 0.8510638 0.9002933 1.0000000
0.8086957 0.8167939 0.8218830 0.8630952 0.8260870 0.8637532 0.8465473 0.8153846 0.8405063 0.7937853 0.8483146 0.8472622 0.8514286 0.8413598 0.8483290 0.8649425 0.7774936 0.8396226 0.8050139 0.8040712 0.8290155 0.8510638 0.8350515 0.8424437 0.8527919 0.8637532 0.8651026 0.8461538 0.8418367 0.8079800 0.8472222 0.8486647 0.8530928 0.8593156 0.8505155 0.8538462 0.8350515 0.8507042 0.8608247 1.0000000 0.8115502 0.8355795 0.8360215 0.8571429 0.8455285 0.8602151 0.8471850 0.8000000 0.8311346 0.8283133 0.8504399 0.8244048 0.8787879 0.8666667 0.8540541 0.8636364 0.7989130 0.8476821 0.8388060 0.8221024 0.8489011 0.8523677 0.8501362 0.8518519 0.8636364 0.8502674 0.8567073 0.8617886 0.8773842 0.8355438 0.8617647 0.8718750 0.8898072 0.8699187 0.8664850 0.8449198 0.8705234 0.8475073 0.8722826 0.8407311 1.0000000 0.8244048 0.8289086 0.8348083 0.8750000 0.8537313 0.8805970 0.8742515 0.8184524 0.8475073 0.8200000 0.8660131 0.8475073 0.8735294 0.8745763 0.8772455 0.8907850 0.7863501 0.8692810 0.8614865 0.8388060 0.8541033 0.8695652 0.8696970 0.8708609 0.8643068 0.8660714 0.8656716 0.8939394 0.8885542 0.8284884 0.8811881 0.8652482 0.8993902 0.8985507 0.8878788 0.8550296 0.8895706 0.8655738 0.8630952 0.8739003 0.8800000 1.0000000 0.8428094 0.8376812 0.8434783 0.8518519 0.8538012 0.8717201 0.8713450 0.8699422 0.8481375 0.8273616 0.8698413 0.8491803 0.8679868 0.8837209 0.8764706 0.8866667 0.8123167 0.8441558 0.8550296 0.8279883 0.8495575 0.8558559 0.8813056 0.8455882 0.8649425 0.8775510 0.8566667 0.8882353 0.8852941 0.8328612 0.8571429 0.8823529 0.8879056 0.8878505 0.8794118 0.8583815 0.9207317 0.8607595 0.8797654 0.8455056 0.8939394 0.9377163 1.0000000
0.4881266 0.5023474 0.4965035 0.5053191 0.4953271 0.5034642 0.5000000 0.4709977 0.4977064 0.4829396 0.5102041 0.4897436 0.5038363 0.4947917 0.5070093 0.4921875 0.4856459 0.5129683 0.5145119 0.4906103 0.4940898 0.5012048 0.5011765 0.4813754 0.4943052 0.4965517 0.5185185 0.5093458 0.5000000 0.4931193 0.5204082 0.5148248 0.5176471 0.4813559 0.5117371 0.4988453 0.5118483 0.5076531 0.5069767 0.5022624 0.5204819 0.5196850 0.5182292 1.0000000 0.5038961 0.5080092 0.5091324 0.5012920 0.5114679 0.5331808 0.5193622 0.4942792 0.5101124 0.4885496 0.5363409 0.5126904 0.5000000 0.5219638 0.5194508 0.5233161 0.4849188 0.5211268 0.5244216 0.5000000 0.5034642 0.5046729 0.5138249 0.4888268 0.5100671 0.5090090 0.5025381 0.5252294 0.5159091 0.4988814 0.5402010 0.5000000 0.5229358 0.5204082 0.5206422 0.5216401 0.5138249 0.5186104 0.5124717 0.5077263 0.5186916 0.5153061 0.5357143 0.5695876 1.0000000 0.3248082 0.3237251 0.3222958 0.3333333 0.3207965 0.3246187 0.3238512 0.3191964 0.3347921 0.3164557 0.3245823 0.3442211 0.3219512 0.3221154 0.3144105 0.3197115 0.3047404 0.3351351 0.3156627 0.3214286 0.3118040 0.3410673 0.3222222 0.3400000 0.3154506 0.3275109 0.3283208 0.3194748 0.3209607 0.3075269 0.3203883 0.3168317 0.3201754 0.3050314 0.3267108 0.3116883 0.3048246 0.3381643 0.3267544 0.3240938 0.3008850 0.3325000 0.3182898 0.3439803 0.3476190 1.00000
1 A B C D E F G H I J K LM NO P Q R S T U VWXY Z a b c d e f g h i j k l m n o p q r s t
2
3
4
5
7
Figure 4.1 A representative AFLP fingerprint of the P. deltoides genotypes generated by the primer combination E-AGC X M-CTC. The lanes ‘A’ to ‘Z’ and ‘a’ to ‘q’ are of the 43 P. deltoides clones and lanes ‘r’, ‘s’ and ‘t’ are of the controls P. ciliata, P. maximowiczii and P. euphratica. The codes for labelling the lanes are same as those given in Table 1. The bands marked are explained in the text.
6
A B CD E F G H I J K L M N OP QR S T U V W XY Z a b c d e f g h i j k l m n o p q r s t
1
2
4
3
5
6
Figure 4.2 A part of DNA profile generated by the AFLP primer combination E-ACC X M-CAG. The lanes ‘A’ to ‘Z’ and ‘a’ to ‘q’ are of the 43 P. deltoides clones and lanes ‘r’, ‘s’ and ‘t’ are of the controls P. ciliata, P. maximowiczii and P. euphratica. The codes for labelling the lanes are same as those given in Table 1. The bands marked are explained in the text.
A B C D E F G H I J K L MN O Q R S P T U V W XY Z a b c d e f g h i j k l m n o p q r s t
1
2
3
4
5
6
Figure 4.3 Autoradiograph of an AFLP fingerprint produced by the primer combination E-AGG X M-CAA. The lanes ‘A’ to ‘Z’ and ‘a’ to ‘q’ are of the 43 P. deltoides clones and lanes ‘r’, ‘s’ and ‘t’ are of the controls P. ciliata, P. maximowiczii and P. euphratica. The codes for labelling the lanes are same as those given in Table 1. The bands marked are explained in the text.
109
3229
23
48
0
20
40
60
80
100
120
No
. o
f M
ark
ers
0.01 to 0.10 0.11 to 0.20 0.21 to 0.30 0.31 to 0.40 0.41 to 0.50
PIC value
Marker frequency
295
115
3527
46
050
100
150
200
250
300
No
. o
f M
ark
ers
0.01 to 0.10 0.11 to 0.20 0.21 to 0.30 0.31 to 0.40 0.41 to 0.50
PIC value
Marker Frequency
Figure 4.4: Polymorphism Information Contents (PIC values) of the AFLP markers for the dataset comprising of the P. deltoides clones along with the outliers (Figure 4A) and the dataset of 43 P. deltoides clones (Figure 4B). The X-axis depicts the five ranges of the PIC values and the Y-axis shows the number of AFLP
A
B
Jaccard's Coefficient
0.32 0.49 0.65 0.81 0.98
126/86 200/86 200/85 L-52/82 L-34/82 L-13 L-75/84 UHF-51 D-121 PD-365-NL 9/54-9-FRG UHF-Pd-90 UHF-60-3 G-48 UHF-14-2 G-3 PD-2498NL A-26/75-FRG G-113324 L-165/84 S7C8 S7C20 IC UHF-100-9 S7C48 26152-NL 2652-NL 3298-NL PD-2094 194(38)-FRG UHF-47A UHF-63 UHF-99 PD-345/183 C-181 65/27 A-50(89) L-181/84 45/51 3295-NL UHF-65-2 L-29/82 UHF-55-2 Pc Pm Pe
Figure 4.5 UPGMA dendrogram based on Jaccard’s similarity coefficient for the 46 Populus genotypes. The three outgroup species P. ciliata, P. maximowiczii and P. euphratica labelled as ‘Pc’, ‘Pm’ and ‘Pe’, respectively, separate out very distinctly from the remaining43 P. deltoides clones. The codes of the genotypes are also given.
A
B
C
D E F
G
Q M c
H
N S L O
K
b
j
l
I
Y
Z m V
X
a
f
e g i
p
U
W k p q
o
H
n
d
T
J
R
P.ciliata P. maximowiczii P.euphratica
0.00 0.24 0.48 0.72 0.96
-0.07
0.13
0.33
0.53
0.74
Figure 4.6A
P.ciliata
P.maximowiczii
P.euphratica
P.deltoides
Figure 4.6 Scatter plot of the P. deltoides clones as generated by Principal Component Analysis in the two datasets i.e. with outgroups P. ciliata, P. maximowiczii and P. euphratica. The positions of the different genotypes are shown through the two-dimension scatter plot in Figure 4.6A and three dimension scatter plot in Figure 4.6B. The codes in the plots are as given in Table 4.1.
P.ciliata
P.maximowiczii
P.euphratica
P.deltoides
Figure 4.6B
0.85 0.88 0.91 0.93 0.96
-0.24
-0.12
0.01
0.13
0.25
126/86
200/86200/85
L-52/82
L-34/82
L-13
L-75/84
L-181/84
L-165/84
L-29/82
G-3
G-48
D-121
UHF-Pd-90
UHF-14-2
UHF-51
UHF-55-2
UHF-60-3UHF-47A
UHF-65-2
UHF-63
UHF-100-9
UHF-99
S7C48
S7C8
S7C20
26152-NL
PD-2498NL
PD-365-NL
3295-NL
3298-NL
2652-NL
PD-2094
9/54-9-FRG
194(38)-FRG
A-26/75-FRG
PD-345/183
G-113324
IC
45/51
A-50(89)
C-181
65/27
65/27
C-181
A-50(89)45/51
IC
G-113324
PD-345/183
A-26/75-FRG
194(38)-FRG
9/54-9-FRG
PD-2094
2652-NL
3298-NL
3295-NL
PD-365-NL
PD-2498NL
26152-NL
S7C20
S7C8
S7C48
UHF-99
UHF-100-9
UHF-63
UHF-65-2
UHF-47AUHF-60-3
UHF-55-2
UHF-51UHF-14-2
UHF-Pd-90
D-121
G-48
G-3
L-29/82
L-165/84
L-181/84
L-75/84
L-13
L-34/82
L-52/82
200/85
200/86
126/86
Figure 4.7A
Figure 4.7 Principal Component Analysis of the P. deltoides clones as generated by NTSYSpc software in the datasets excluding outgroup species. Figure 4.7A and 4.7B depict the 43 P. deltoides species positions in two dimensions and three dimensions respectively. The codes are as given in Table 4.1.
Figure 4.7B
+---------A
+-62.0
| | +----B
| +100.0
+----------------------7.4 +----C
| |
| | +----S
| +------25.0
| +----Q
|
| +----D
| +---------------------22.4
| | +----E
| |
| | +----F
| | +-80.2
| | +-35.0 +----G
| | | |
| | +--1.8 +---------L
| | | |
| | | | +---------l
| | | +--9.2
+--4.8 | | | +----b
| | +--0.8 | +-68.2
| | | | +--0.2 +----K
| | | | | |
| | | | | | +---------I
| | | | | | +-33.2
| | | | | | | | +----M
| | | | | | | +-39.4
| | | | | +-21.0 +----Y
| | | | | |
| | | | | | +----m
| | | | | +------56.4
| | | | | +----Z
| | | | |
| | | +--0.2 +---------j
| | | | +-42.2
| | | | | | +----h
| | | +-------6.0 +-81.8
| | | | | +----c
| +--1.8 | |
| | | +--------------X
| | |
| | | +---------O
+--4.8 | | +-36.0
| | | | | | +----P
| | | | | +-48.2
| | | +-------0.6 +----N
| | | |
| | | | +----W
| | | | +-45.8
| | | +-25.6 +----U
| | | |
| | | +---------V
| | |
| | | +---------i
| | | +-99.2
| | | | | +----g
| | | | +-74.8
+--6.8 | +----------------46.2 +----e
| | | |
| | | | +----f
| | | +------91.6
| | | +----a
| | |
| | | +-------------------n
| | | |
| | +---------------------17.2 +--------------o
+-15.0 | | |
| | | +-69.8 +----q
| | | | +-98.6
| | | +-76.8 +----p
| | | |
| | | +---------k
| | |
+100.0 | +-------------------------------------------------H
| | |
| | | +----T
| | +----------------------------------------------22.0
| | +----d
+100.0 |
| | | +----R
| | +---------------------------------------------------36.8
| | +----J
| |
| | +----s
| +-------------------------------------------------------100.0
| +----r
|
+---------------------------------------------------------------------t
Figure 4.8 Bootstrap tree of the 46 genotypes showing the bootstrap values at each node of the tree obtained by 500 replications.