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


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



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



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



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



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



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



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 &



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



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



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.



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



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.

study of populus deltoides genetic...

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