The family Salicaceae includes three genera, Salix, Populus and
Chosenia with more than 200 species in the world (Rehder,
1940). Salix is most abundant in the temperate regions of Northern
Hemisphere. Most of the Kashmir Salix species have been introduced and
planted from cuttings nearly all of which are represented by male plants, which
make identification more difficult. The various Salix species were introduced
primarily for wicker works and sports goods in the valley. Cricket bats are
obtained from S. alba L. sp. coerulea (Sm.) Rech. and S. fragilis
L. The important wicker willows are S. triandra L., S. purpurea
L. and S. viminalis L.; from the twigs of these plants the baskets are
made (Javeid, 1972). The bark and leaves of Salix
also contain substantial amounts of salicylic acid, which has antipyretic and
The documented history of Salix culture in North America started during
the period from 1840-1850. The importance of selecting clones with desirable
form and wood qualities that were adapted to local soils and pests was recognized
in the earliest days of willow culture (Hubbard, 1904).
Growing willows (Salix species) in intensive culture systems for biomass
can be used for energy or conversion to high value products. It is gaining increasing
worldwide interest. Willows in general are perennial, out-crossing, insect pollinated
species with a long life history and overlapping generations, all contributing
to a relatively high degree of heterozygosity and intra- as well as inter-population
genetic variation (Kopp et al., 2002). Interest
in growing and using willows for energy and to produce high-value chemicals
and other bio-based products increased in the United States during the past
two decades (Abrahamson et al., 1990).
Modern techniques, such as Amplified Fragment Length Polymorphism (AFLP) is
a genetic fingerprinting technique that may be useful in predicting the likelihood
that parental combinations will yield highly variable progeny. AFLP has been
effectively used to fingerprint willows for genetic distance estimates and clone
identity verification (Barker et al., 1999) and
to identify willow hybrids in natural stands (Beismann
et al., 1997).
The AFLP approach, which enables simultaneous analysis of a large number of marker loci throughout the genome, appears to be remarkably powerful. The objective of this work is to study the genetic relationships among different S. viminalis cultivars through AFLP markers.
MATERIALS AND METHODS
Plant material: For the study entitled Genetic Diversity in Salix
viminalis in Kashmir Valley, India, a total of 4 accessions of Salix
viminalis were collected in the year 2005 from the valley for evaluation
of genetic diversity (Fig. 1). The samples studied are cultivated
in different regions of the valley. These samples were also being cultivated
in the Kashmir University Botanical Garden.
||General morphology of Salix viminalis species/cultivars
in Kashmir Valley; (A) Salix viminalis, (B) Salix viminalis
(white) , (C) Salix viminalis (red) and (D) Salix viminalis
The analysis was carried out in order to estimate the genetic variation within the different cultivars of Salix viminalis.
DNA isolation: Young Salix leaves were collected and lyophilized (in liquid nitrogen at -196°C) over a period of 10-15 days for DNA extraction. The genomic DNA was extracted from 70-75 mg of the freeze dried leaf sample using QIAGEN Dneasy Plant Mini Kit (QIAGEN GmbH. Max-Volmerstrasse 4, 40724 Hilden, Germany). Lyophilized tissue was ground to a fine powder in pestle and mortar using liquid nitrogen. Ground tissue was transferred to an appropriately sized centrifuge tube to which 400 μL of lysis buffer (1% cetyltrimethyl ammonium bromide (CTAB), 5% polyvinyl pyrrolidone (PVP), 1.4 M NaCl, 20 mM EDTA, 10 mM Tris-HCl (pH-8.0) and 350 mM 2-mercaptoethanol) and 4 μL of RNase stock solution was added. Samples were mixed thoroughly by vortexing and were incubated for 10 min. at 65°C. 150 μL of precipitation buffer was added to each tube and again incubated for 10 min at -20°C. The lysate was then centrifuged at 15000 rpm for 5 min. After centrifugation the supernatant was transferred to the QIA shredder spin column and centrifugation was carried out at 15000 rpm for 2 min. Flow through fraction from the above step was transferred to a new tube without disturbing the cell debris pellet and 400 μL of precipitation buffer was added to the lysate and mixed gently. The mixture was then transferred to mini spin columns (binding columns) and centrifuged for 1 min at 8000 rpm. The flow through fraction was discarded and to the retained fraction 500 μL of wash buffer (70% ethanol) was added and centrifuged again at 8000 rpm for 1 min. Ten microliter of elution buffer was then added and incubated for 5 min at room temperature. Centrifugation at 8000 rpm for 1 min was done to elute. DNA concentration was estimated by comparison to serial dilution of a lambda DNA standard in a 1.0% agarose gel (Fig. 2).
AFLP analysis: AFLP analysis was performed according to procedure described
by Vos et al. (1995), with a commercially available
kit (AFLP®Analysis System I, Invitrogen Life Technologies, Carlsbad, CA).
Approximately 200 ng DNA of each sample was digested with Eco R1/Mse1 restriction
||Isolated DNA from 4 Salix viminalis species/cultivars
resolved on 1.0% agarose. L represents Lambda phage DNA
After the ligation of the digested DNA, the reaction mixture was diluted 10
fold with TE buffer containing 10 mM Tris-HCL, pH 8.0, 0.1 mM EDTA. The number
of cycles for pre-amplification reaction and selective amplification reaction
was 20 and 23 cycles, respectively. The pre-amplification product was also diluted
1:50. The amplification products were separated on 6% (w/v) polyacrylamide gels
containing 29:1 acrylamide : Bis-acryl-amide (Fisher Scientific, Chicago, IL),
7.5 M urea and 1x TBE buffer (1, 1, 2, 2-tetra bromoethane) used with 0.4 mm
spacers and a shark-tooth comb. The gels were electrophoresed for about 20 min.
with 1x TBE. Electrophoresis was performed at constant temperature and wattage
(45-50°C, 100 w) for about 2.5 h. The gels were stained by silver staining
process and the bands were visualized with a transilluminator (Fisher Scientific,
Chicago, IL) (Fig. 3A, B and 4A,
||AFLP fingerprints of four Salix viminalis species/cultivars
using primers (A) 73 and (B) 74. M represents 1 kb DNA marker
||AFLP fingerprints of four Salix viminalis species/cultivars
using primers (A) 75 and (B) 76. M represents 1 kb DNA marker
||Dendrogram showing diversity in Salix viminalis species/cultivars
in Kashmir valley
Data analysis: Polymorphic AFLP markers were manually scored as binary data with presence as 1 and absence as 0. Monomorphic markers were not scored. The data was compiled by NTSYSPC (Numerical Taxonomy and multivariate analysis System) software version 2.0 (Exeter Software, New York). Similarity matrix was computed by Jaccards coefficient. This analysis was also used for phylogenetic tree estimation, which was visualized as a graphical dendrogram. The resulting dendrogram provides a good estimate of the phylogeny of a particular group of organisms (Fig. 5).
The summary of AFLP markers produced by four primer pairs across all genotypes is given in Table 1. The four primers generated a total of 240 bands of which 197 (82%) were polymorphic over all the genotypes. The capability of different primers to generate polymorphic AFLP markers varied significantly, ranging from 40-55 polymorphic bands per primer over all the genotypes. Thereby it confirms the high multiplex ratio expected from the AFLP technique.
Similarity index was estimated using the Jaccard coefficient which revealed accessions SL-11 (Salix viminalis white cultivar) and SL-12(Salix viminalis red cultivar) sharing a similarity value of 0.69 which is highest among the four accessions studied. Accessions SL-6 (Salix viminalis) and SL-25 (Salix viminalis black cultivar) are the most diverse genotypes as revealed by the AFLP analysis showing a similarity value of 0.37. Based on genetic similarity values, a phenetic dendrogram was constructed using UPGMA (Unweighed Pair Group Method Arithmetic mean) analysis. On examination of dendrogram, it is clear that there is a large genetic variation. Accessions SL-11 and SL-12 show genetic similarity value of 0.69 while the comparison of accession SL-25 with SL-11 and SL-12 show the genetic similarity value of about 0.40 (Table 2).
AFLP results mostly confirmed that the four Salix genotypes studied are distinct from one another as is evident from the large genetic distances among them. However, as revealed from the study that morphologically there is lot of similarity but when these cultivars were analyzed using AFLP technique there was found difference among these cultivars.
Molecular fingerprint data have been used to identify particular parent combinations
that are likely to yield desirable clones based on maximizing the amount of
molecular diversity among crossed genotypes (Kopp et
Results of AFLP analysis in this study suggest that there was in general, large
variability among Salix viminalis cultivars. One hundred and ninty seven
polymorphic loci were detected in the four S. viminalis cultivars using
only four AFLP primer pairs. Eighty two percent of the fragment types were polymorphic.
Every cultivar could be distinguished with either primer pair alone.
||List of AFLP primers used in DNA fingerprinting of Salix
viminalis cultivars in Kashmir valley
||Similarity matrix of Salix viminalis cultivars in Kashmir
These results were similar to those reported for clones of Salix viminalis
and its hybrids grown for bio-energy in Europe, where AFLP analysis of 29 willow
clones with five AFLP primer pairs yielded 919 different fragment types, of
which 752 (81.8%) were polymorphic (Barker et al.,
1999). The high percentage of polymorphic bands detected in willows in AFLP
experiments reflects the low level of domestication in this genus.
Although, the number of polymorphic bands used to estimate similarity among
Salix viminalis cultivars was relatively small in this study, there appears
to be sufficient polymorphism to provide reliable estimate of genetic diversity.
In a study by Zhu et al. (1998) about 82 to 140
polymorphic AFLP markers were sufficient to accurately estimate similarities
among rice accessions. Similarly, Pejic et al. (1998)
obtained genetic similarity between inbred maize lines which was most accurately
estimated with only 150 polymorphic AFLP bands.
The number of polymorphic bands necessary to accurately estimate similarity
among willows should be relatively low because of the broad geographic range
from which parents were selected and the high degree of genetic variation that
exists in this species (Kopp et al., 2002).
Pejic et al. (1998) reported that 150 polymorphic
bands make it possible for a researcher to reliably estimate genetic similarities
among genotypes within the same species. In confirmation to this, it was found
that with four primer pairs, generating 197 polymorphic bands, it was possible
to fingerprint all of the four species/varieties/cultivars included in this
Large molecular genetic variation was observed in the current study because
it includes outcrossing species and maintains large genetic variation within
populations and high individual-tree heterozygosity. The same results were also
reported by Aravanopoulos et al. (1998). Further,
genus Salix has a widespread geographic distribution, widely dispersed
seeds and both sexual and asexual reproduction which are characteristics that
tend to be associated with large genetic diversity (Hamrick
et al., 1992).
The number of polymorphic bands necessary to accurately discriminate among
individuals is determined by the degree of relatedness among individuals being
compared, with distantly related individuals requiring relatively few bands,
confirming the studies carried out by Tivang et al.
The authors are highly thankful to Director, Ankur Seeds Pvt. Ltd. Nagpur, who provided all the necessary facilities for carrying out the study.