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ESTIMATION OF GENETIC DIVERSITY IN PROGENIES OF SELECTED GENOTYPES OF ULMUS VILLOSA BRANDIS USING RAPD MARKERS

SAPNA THAKUR, I. K. THAKUR, N. B. SINGH, J. P. SHARMA AND M. SANKANUR

Department of Tree Improvement and Genetic Resources, College of ForestryDr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan- 173 230 Himachal Pradesh, INDIA

email: ikuhf@rediffmail.com

ABSTRACT

Molecular diversity among 23 promising progenies of Ulmus villosa, which were raised from the seeds collected from various seed sources in Himachal Pradesh (India), was estimated using 10 RAPD primers. A total of 57 markers were generated, all of the 10 primers showed 100 per cent polymorphism. The similarity coefficient among 23 progenies of U. villosa ranged from 0.00 to 0.70. In which, progeny Jugahan-T was found to be the most divergent which separated 3

itself from rest of the progenies at similarity value (0.04) and could be used as a parent in hybridization programme and further improvement programmes. The progenies were grouped into 4 clusters. The cluster II consisted maximum of 12 progenies followed by cluster III (5 progenies), cluster IV (4 progenies) whereas cluster I consisted of single progeny. RAPD analysis proved helpful for estimating the magnitude of genetic diversity at molecular level.

Key words: RAPD, Ulmus villosa, Progenies, Genetic diversity.

Studies on molecular diversity among 23 promising progenies of Ulmus villosa revealed Jugahan –T 3

to be the most divergent and could be used as a parent in hybridization programme.

Introduction considered as one of the most important agro-forestry trees in the Kashmir region. It also has a great potential The elms (Ulmus L.) are represented by outside its natural range for use on degraded land (Singh, approximately 35 species distributed throughout the 1982; Bhardwaj and Mishra, 2005). However, the Dutch temperate regions of the Northern Hemisphere and into elm disease (Kalas et al., 2006), caused by certain fungi the subtropics of Central America and Southeast Asia, (Ophiostoma spp), is one of the most serious diseases including six species in eastern North America (Pooler known to trees, has ravaged elm populations all over and Townsend, 2005). There are five species of Ulmus Europe. This poses a challenge for future conservation of found in India, four namely U. wallichiana, U. villosa, U. elm, which in turn necessitates more knowledge about pumila and U. chumlia from N.W Himalaya and U. the distribution of variation in adaptive traits in the lanceifolia from north-eastern regions of the country. species. Apart from this the species being one of the cold Himalayan elms are the source of best fodder and quality hardy and disease resistant species of elm, has been timber. U. wallichiana is lopped for fodder which causes introduced in Europe and North America as an the depletion of regeneration. It is already categorized as ornamental tree and for breeding purposes. Some clones v u l n e r a b l e s p e c i e s i n R e d D a t a B o o k of this species have proved resistant against Dutch elm (www.iucnredlist.org). Ulmus villosa Brandis, commonly disease (DED) that can be used in breeding programmes known as marinoo in India, is a small or medium sized for the development of disease resistant hybrids deciduous tree belonging to family Ulmaceae (Melville (Santamour, 1979). and Heybroek, 1971). It is one of the more distinctive

Asiatic elms and a species capable of remarkable Molecular techniques have been found to be more longevity (Singh, 1991). It grows up to 20-30 m in height useful and accurate for determination of both at elevations from 1200 m to 2500 m with a scattered interspecific and intraspecific genetic variation in plants. distribution in the north western Himalayas. DNA markers can be used early in tree growth and

development to predict dissimilar genetic backgrounds The seed viability is high but seed longevity is low. and to determine which traits a particular individual is It finds greater favour on account of its multiplicity of carrying by examining these small segments. Markers can uses and fast growth habit. It is a multipurpose discern between single or multi-locus modifications as agroforestry tree species producing fodder, fuel and well as dominant or co-dominant alterations in a single timber. Inspite of its immense popularity and multiplicity individual. When applied to the conservation and of its uses less attention has been paid on improvement breeding of fine hardwoods many diverse DNA marker of this species (Melville and Heybroek, 1971). It is

Indian Forester, 140 (12) : 1221-1229, 2014http://www.indianforester.co.in

ISSN No. 0019-4816 (Print)ISSN No. 2321-094X (Online)

Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12232014]

system types have been utilized. DNA markers allow for Ulmus americana clones. more accurate determination of the region of origin for a There is no evidence regarding molecular particular tree and detection of specific gene flow events. characterization of the species. Thus the aim of this work The many DNA marker techniques are similar in that was to study genetic diversity in progenies of selected these can be used even where there is a single nucleotide genotypes in Ulmus villosa Brandis using RAPD markers change in a gene or tandem DNA repeat. Unlike and to assess conservation strategies for the species. In morphological markers these changes are not apparent the present investigation RAPD markers has been used in the phenotype of the individual and are often for assay in genetic variation at molecular level of 23 insignificant in its physiological development. Molecular progenies to select the best genotypes on the basis of markers has been used to understand hybridization and their progeny performance. This may be perhaps the first species differentiation in forest trees such as Acer (Joung scientific paper to deal with the study of genetic diversity et al., 2001; Skepner and Krane, 1998), Betula (Palme et in progenies of selected genotypes in U. villosa which al., 2004; Anamthawat-Jonsson and Thorsson, 2003), were collected from various seed sources of the state of Liriodendrone (Li and Wang, 2002), Platanus (Vigouroux Himachal Pradesh in India.et al., 1997), Fagus (Ohyama et al., 1999; Gailing and Von

Material and MethodsWuelisch, 2004), Tilia (Fineschi et al., 2003), Olea (Claros

Plant materialet al., 2000; Fabri et al., 1995), Ficus (Hadia et al., 2008). Mylett et al. (2007) reported that RAPDs were used to Well matured seeds were collected from five indicate genetic variability between individual Tilia mother trees (15-25cm DBH) each at six sites viz., S -Jadh 1

cordata Mill. clusters within the same woodland and (800m), S -Jugahan (800m), S -Jhidi (1089m) forest area 2 3

surrounding areas. In F. excelsior (Pvingila et al., 2005) it in Mandi district and S -Jagoti (1824m), S -Katouch 4 5

was noted that the unique RAPD phenotype, the basis for (1900m) and S -Andhra (2200m) area from Pabbar valley 6

individual tree identification, indicated that RAPD in Shimla district of Himachal Pradesh. The progenies markers can indeed confirm origin of a given forest tree. were then raised under nursery conditions in the A simple, efficient and genetically stable method for P. experimental field. Twenty three best performing occidentalis was recently presented using RAPDs by Sun progenies (Table 1) were selected for the studies.et al. (2009) and only with RAPD in Dalbergia sissoo (Rout

Collection of plant material and genomic DNA extractionet al., 2003) was done. Variation in Melientha suavis was Fresh and disease free young leaves were detected with RAPD (Prathepha, 2000). It is also used as

collected from these selected progenies for molecular a Randomly Amplified Polymorphic DNA (RAPD) markers, in particular, have been successfully employed for determination of intraspecies genetic diversity in several plants (Li et al., 2008; Goodall-Copestake et al., 2005; Nanda et al., 2004; Amri and Mamboya, 2012), sex determination in dioceous tree Salix viminalis (Alstrom-Rapaport et al., 1998). Random amplified polymorphic DNA (RAPD) analysis has proved useful for estimating genetic diversity particularly to assist in the conservation of rare species and plant genetic resources (Anderson and Fairbanks, 1990). The use of dominant markers to assess genetic variability between individuals and populations is promising because many polymorphic loci can be obtained fairly easily, in a relatively short time and at low cost, without any prior knowledge of the genome of the species under study (Nybom and Bartish, 2000; Nybom, 2004). RAPD analysis in particular has proven to be a rapid and efficient means of genome mapping (Williams et al., 1990) and have been successfully used for differentiating species of a genus based on their similarities and geographical proximities (Thomas et al., 2001). Pooler and Townsend (2005) indicated success with AFLP in determining geographic origins and genetic distances between selections of Ulmus laevis Palli and

variability studies and carried to the laboratory in brown paper bags within 2-3 hours of collection and kept in deep freezer (- 20°C) for further DNA extraction. Fresh leaf tissue (~ 0.5 g) was crushed in 7 ml extraction buffer (10 per cent (w/v) CTAB (N-cetyle, N, N-trimethyle- ammonium bromide), 0.5M EDTA (pH 8.0), 5M NaCl and

01M Tris pH 8.0. The powder was either stored at -40 C or used for DNA isolation immediately. Total genomic DNA was isolated using the Doyle and Doyle (1987) method with slight modification made in buffer concentrations. The quality of DNA was tested on 0.8% agarose gel and quantification was done using Perkin Elmer UV/VIS spectrophotometer and diluted to 5ng/µl for further PCR (Polymerase chain reaction) amplification using CR Corbett thermocycler.

PCR amplification and electrophoresis

Ten decamer primers were used for the current study (Table 2). DNA was amplified by PCR amplification Results reaction. The 25µl of reaction mixture contained 20ng of RAPD banding patternDNA, 0.75 units of Taq DNA polymerase, 2.5µl of 10X Taq

Each primer generated a unique set of buffer (50mM MgCl , 10mM Tris-Cl), 1.25µl of pooled 2 amplification products revealing polymorphism and high dNTP's (2.5mM each) and 10ng of primer. PCR conditions

levels of genetic diversity among different progenies. The used for RAPD amplification included initial denaturation

number of bands recognized by the software, Alpha 0for 3 min at 94 C followed by 45 cycles of amplification Imager for each primer ranged from 3 (OPA-04) to 8 (0PA-0(denaturation at 92 C for 45 seconds, annealing of primer 07) (Table 2). All the ten primers used in this analysis 0 0at 36 C for 1min and primer amplification at 72 C for 2 yielded a total of 57 scorable bands with an average of 0min) and final extension at 72 C for 10 min. 5.70 bands per primer. All the scorable bands showed

Amplification products stained in ethidium polymorphism resulting in 100 % polymorphism among bromide were separated on 2 per cent agarose gel using 23 progenies (Tables 2 and 3). The banding pattern 1X TBE buffer (Tris HCI pH 8.0, Boric Acid, Ethylene generated by each RAPD primer for 23 progenies is diamine-tetra acetic acid) on horizontal gel presented in Table 4. The mean coefficient value of any electrophoresis apparatus and photographed in Alpha progeny gave an idea about its overall relatedness with all Imager gel documentation system. 1kb and 100bp DNA other progenies in the study. The coefficient values mass ladder were used as molecular weight markers in ranged from 0.04 to 0.70 (Table 6). This indicated a fair first and last well of respective gel. Data analysis and range of variability in the similarity coefficient values clustering was done by UPGMA using SAHN module of suggesting a broad genetic base of 23 progenies included NTSYSpc. Version 2.02e (Rohlf, 1998). in the experiment. Maximum similarity (70%) was

Table 1: Details of Ulmus villosa 23 progenies used in RAPD studies

S. no. Progenies code Seed source District 1. Jh-T2 Jhidi, Tree no. 2 Mandi 2. Jh-T3 Jhidi, Tree no. 3 3. Jh-T5 Jhidi, Tree no. 5 4. Ju-T1 Jugahan, Tree no. 1 5. Ju-T2 Jugahan, Tree no. 2 6. Ju-T3 Jugahan, Tree no. 3 7. Ju-T4 Jugahan, Tree no. 4 8. Ju-T5 Jugahan, Tree no. 5 9. Ja-T1 Jadh, Tree no. 1

10. Ja-T2 Jadh, Tree no. 2 11. Ja-T3 Jadh, Tree no. 3 12. Ja-T1 Jadh, Tree no. 4 13. Ja-T1 Jadh, Tree no. 5 14. Ka-T1 Katouch, Tree no.1 Shimla 15. Ka-T2 Katouch, Tree no.2 16. Ka-T3 Katouch, Tree no.3 17. Ka-T4 Katouch, Tree no.4 18. Ka-T5 Katouch, Tree no.5 19. Jag-T1 Jagoti, Tree no.1 20. Jag-T2 Jagoti, Tree no.2 21. Jag-T3 Jagoti, Tree no.3 22. Jag-T4 Jagoti, Tree no.4 23. Jag-T5 Jagoti, Tree no.5

S. No Parameters Remarks 1 Total number of primers examined 10 2 Total number of polymorphic primers 10 3 Total number of bands amplified from

polymorphic primers 57

4 Total number of polymorphic bands identified

57

5 Total number of monomorphic bands 0 6 Average number of polymorphic bands

per primer 5.70

7 Per cent of total polymorphic bands 100% 8 Number of primers exhibited 100 %

polymorphism 10

9 Size range of RAPD markers 147.10 bp to 2901.97 bp

10 Number of amplification products per primer

3 (OPA - 04) to 8 (OPA - 07)

Table 2: Summary of RAPD amplified products obtained from 23 progeny of Ulmus villosa

Table 3: Total numbers of amplified and polymorphic fragments generated by PCR using RAPD primers

S. No Primer name

Total no. of scorable bands

Total no. of polymorphic

bands

Total no. of monomorphic

bands

Polymorphism (%)

100Xy

x

Size range of amplified products

(bp)

1 OPA-01 6 6 0 100 147.10 - 1082.65 2 OPA- 04 3 3 0 100 151.83 - 246.77 3 0PA- 05 5 5 0 100 593.75 - 2901.97 4 OPA- 07 8 8 0 100 156.91 - 2050.97 5 OPA- 09 6 6 0 100 187.72 - 957.76 6 OPC- 08 7 7 0 100 226.70 - 874.90 7 OPC- 11 4 4 0 100 203.07 - 584.67

8 OPB-11 6 6 0 100 182.11 - 2086.89

9 OPL- 06 6 6 0 100 278.63 - 814.73 10 OPS- 15 6 6 0 100 183.98 - 2027.10

TOTAL 57 57 0 100 147.10 - 2901.97

1222 The Indian Forester [December

Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12232014]

system types have been utilized. DNA markers allow for Ulmus americana clones. more accurate determination of the region of origin for a There is no evidence regarding molecular particular tree and detection of specific gene flow events. characterization of the species. Thus the aim of this work The many DNA marker techniques are similar in that was to study genetic diversity in progenies of selected these can be used even where there is a single nucleotide genotypes in Ulmus villosa Brandis using RAPD markers change in a gene or tandem DNA repeat. Unlike and to assess conservation strategies for the species. In morphological markers these changes are not apparent the present investigation RAPD markers has been used in the phenotype of the individual and are often for assay in genetic variation at molecular level of 23 insignificant in its physiological development. Molecular progenies to select the best genotypes on the basis of markers has been used to understand hybridization and their progeny performance. This may be perhaps the first species differentiation in forest trees such as Acer (Joung scientific paper to deal with the study of genetic diversity et al., 2001; Skepner and Krane, 1998), Betula (Palme et in progenies of selected genotypes in U. villosa which al., 2004; Anamthawat-Jonsson and Thorsson, 2003), were collected from various seed sources of the state of Liriodendrone (Li and Wang, 2002), Platanus (Vigouroux Himachal Pradesh in India.et al., 1997), Fagus (Ohyama et al., 1999; Gailing and Von

Material and MethodsWuelisch, 2004), Tilia (Fineschi et al., 2003), Olea (Claros

Plant materialet al., 2000; Fabri et al., 1995), Ficus (Hadia et al., 2008). Mylett et al. (2007) reported that RAPDs were used to Well matured seeds were collected from five indicate genetic variability between individual Tilia mother trees (15-25cm DBH) each at six sites viz., S -Jadh 1

cordata Mill. clusters within the same woodland and (800m), S -Jugahan (800m), S -Jhidi (1089m) forest area 2 3

surrounding areas. In F. excelsior (Pvingila et al., 2005) it in Mandi district and S -Jagoti (1824m), S -Katouch 4 5

was noted that the unique RAPD phenotype, the basis for (1900m) and S -Andhra (2200m) area from Pabbar valley 6

individual tree identification, indicated that RAPD in Shimla district of Himachal Pradesh. The progenies markers can indeed confirm origin of a given forest tree. were then raised under nursery conditions in the A simple, efficient and genetically stable method for P. experimental field. Twenty three best performing occidentalis was recently presented using RAPDs by Sun progenies (Table 1) were selected for the studies.et al. (2009) and only with RAPD in Dalbergia sissoo (Rout

Collection of plant material and genomic DNA extractionet al., 2003) was done. Variation in Melientha suavis was Fresh and disease free young leaves were detected with RAPD (Prathepha, 2000). It is also used as

collected from these selected progenies for molecular a Randomly Amplified Polymorphic DNA (RAPD) markers, in particular, have been successfully employed for determination of intraspecies genetic diversity in several plants (Li et al., 2008; Goodall-Copestake et al., 2005; Nanda et al., 2004; Amri and Mamboya, 2012), sex determination in dioceous tree Salix viminalis (Alstrom-Rapaport et al., 1998). Random amplified polymorphic DNA (RAPD) analysis has proved useful for estimating genetic diversity particularly to assist in the conservation of rare species and plant genetic resources (Anderson and Fairbanks, 1990). The use of dominant markers to assess genetic variability between individuals and populations is promising because many polymorphic loci can be obtained fairly easily, in a relatively short time and at low cost, without any prior knowledge of the genome of the species under study (Nybom and Bartish, 2000; Nybom, 2004). RAPD analysis in particular has proven to be a rapid and efficient means of genome mapping (Williams et al., 1990) and have been successfully used for differentiating species of a genus based on their similarities and geographical proximities (Thomas et al., 2001). Pooler and Townsend (2005) indicated success with AFLP in determining geographic origins and genetic distances between selections of Ulmus laevis Palli and

variability studies and carried to the laboratory in brown paper bags within 2-3 hours of collection and kept in deep freezer (- 20°C) for further DNA extraction. Fresh leaf tissue (~ 0.5 g) was crushed in 7 ml extraction buffer (10 per cent (w/v) CTAB (N-cetyle, N, N-trimethyle- ammonium bromide), 0.5M EDTA (pH 8.0), 5M NaCl and

01M Tris pH 8.0. The powder was either stored at -40 C or used for DNA isolation immediately. Total genomic DNA was isolated using the Doyle and Doyle (1987) method with slight modification made in buffer concentrations. The quality of DNA was tested on 0.8% agarose gel and quantification was done using Perkin Elmer UV/VIS spectrophotometer and diluted to 5ng/µl for further PCR (Polymerase chain reaction) amplification using CR Corbett thermocycler.

PCR amplification and electrophoresis

Ten decamer primers were used for the current study (Table 2). DNA was amplified by PCR amplification Results reaction. The 25µl of reaction mixture contained 20ng of RAPD banding patternDNA, 0.75 units of Taq DNA polymerase, 2.5µl of 10X Taq

Each primer generated a unique set of buffer (50mM MgCl , 10mM Tris-Cl), 1.25µl of pooled 2 amplification products revealing polymorphism and high dNTP's (2.5mM each) and 10ng of primer. PCR conditions

levels of genetic diversity among different progenies. The used for RAPD amplification included initial denaturation

number of bands recognized by the software, Alpha 0for 3 min at 94 C followed by 45 cycles of amplification Imager for each primer ranged from 3 (OPA-04) to 8 (0PA-0(denaturation at 92 C for 45 seconds, annealing of primer 07) (Table 2). All the ten primers used in this analysis 0 0at 36 C for 1min and primer amplification at 72 C for 2 yielded a total of 57 scorable bands with an average of 0min) and final extension at 72 C for 10 min. 5.70 bands per primer. All the scorable bands showed

Amplification products stained in ethidium polymorphism resulting in 100 % polymorphism among bromide were separated on 2 per cent agarose gel using 23 progenies (Tables 2 and 3). The banding pattern 1X TBE buffer (Tris HCI pH 8.0, Boric Acid, Ethylene generated by each RAPD primer for 23 progenies is diamine-tetra acetic acid) on horizontal gel presented in Table 4. The mean coefficient value of any electrophoresis apparatus and photographed in Alpha progeny gave an idea about its overall relatedness with all Imager gel documentation system. 1kb and 100bp DNA other progenies in the study. The coefficient values mass ladder were used as molecular weight markers in ranged from 0.04 to 0.70 (Table 6). This indicated a fair first and last well of respective gel. Data analysis and range of variability in the similarity coefficient values clustering was done by UPGMA using SAHN module of suggesting a broad genetic base of 23 progenies included NTSYSpc. Version 2.02e (Rohlf, 1998). in the experiment. Maximum similarity (70%) was

Table 1: Details of Ulmus villosa 23 progenies used in RAPD studies

S. no. Progenies code Seed source District 1. Jh-T2 Jhidi, Tree no. 2 Mandi 2. Jh-T3 Jhidi, Tree no. 3 3. Jh-T5 Jhidi, Tree no. 5 4. Ju-T1 Jugahan, Tree no. 1 5. Ju-T2 Jugahan, Tree no. 2 6. Ju-T3 Jugahan, Tree no. 3 7. Ju-T4 Jugahan, Tree no. 4 8. Ju-T5 Jugahan, Tree no. 5 9. Ja-T1 Jadh, Tree no. 1

10. Ja-T2 Jadh, Tree no. 2 11. Ja-T3 Jadh, Tree no. 3 12. Ja-T1 Jadh, Tree no. 4 13. Ja-T1 Jadh, Tree no. 5 14. Ka-T1 Katouch, Tree no.1 Shimla 15. Ka-T2 Katouch, Tree no.2 16. Ka-T3 Katouch, Tree no.3 17. Ka-T4 Katouch, Tree no.4 18. Ka-T5 Katouch, Tree no.5 19. Jag-T1 Jagoti, Tree no.1 20. Jag-T2 Jagoti, Tree no.2 21. Jag-T3 Jagoti, Tree no.3 22. Jag-T4 Jagoti, Tree no.4 23. Jag-T5 Jagoti, Tree no.5

S. No Parameters Remarks 1 Total number of primers examined 10 2 Total number of polymorphic primers 10 3 Total number of bands amplified from

polymorphic primers 57

4 Total number of polymorphic bands identified

57

5 Total number of monomorphic bands 0 6 Average number of polymorphic bands

per primer 5.70

7 Per cent of total polymorphic bands 100% 8 Number of primers exhibited 100 %

polymorphism 10

9 Size range of RAPD markers 147.10 bp to 2901.97 bp

10 Number of amplification products per primer

3 (OPA - 04) to 8 (OPA - 07)

Table 2: Summary of RAPD amplified products obtained from 23 progeny of Ulmus villosa

Table 3: Total numbers of amplified and polymorphic fragments generated by PCR using RAPD primers

S. No Primer name

Total no. of scorable bands

Total no. of polymorphic

bands

Total no. of monomorphic

bands

Polymorphism (%)

100Xy

x

Size range of amplified products

(bp)

1 OPA-01 6 6 0 100 147.10 - 1082.65 2 OPA- 04 3 3 0 100 151.83 - 246.77 3 0PA- 05 5 5 0 100 593.75 - 2901.97 4 OPA- 07 8 8 0 100 156.91 - 2050.97 5 OPA- 09 6 6 0 100 187.72 - 957.76 6 OPC- 08 7 7 0 100 226.70 - 874.90 7 OPC- 11 4 4 0 100 203.07 - 584.67

8 OPB-11 6 6 0 100 182.11 - 2086.89

9 OPL- 06 6 6 0 100 278.63 - 814.73 10 OPS- 15 6 6 0 100 183.98 - 2027.10

TOTAL 57 57 0 100 147.10 - 2901.97

1222 The Indian Forester [December

Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12252014]1224 The Indian Forester [December

observed between Jag-T and Jag-T . However, the Jhidi-T , Jadh-T , Jhidi-T and Katouch-T and showing 43 2 3 1 5 5 4

minimum similarity coefficient values (0.00) were per cent similarity with the rest of the progenies.observed in few progenies. The inference of Table 5 Discussion shows the informative RAPD markers specific for a

Molecular techniques have been found to be more particular progeny. Primer OPA-05 and OPA-07 produced

useful and accurate for determination of both unique bands of size approximately 708.20 bp and

interspecific and intraspecific genetic variation in plants. 156.91 bp for the JuT (Jugahan), respectively. These 1 Randomly amplified polymorphic DNA (RAPD) markers, informative primers producing unique bands for a

in particular, have been successfully employed for particular progeny can be specifically used for the study

determination of intraspecies genetic diversity in several of particular genotype.

plants. RAPD analysis has been successfully used for Cluster analysis differentiating species of a genus based on their

similarities and geographical proximities (Thomas et al., Dendrogram was created using the similarity 2001). In the present investigation RAPD markers used coefficient and un-weighted pair group method with for assay in genetic variation at molecular level showed arithmetic average (UPGMA) (Fig. 1). A critical perusal of that RAPDs were informative for revealing relationship dendrogram reveals that the distribution of various based upon similarity with in reference set of progenies.progenies into clusters and within cluster was random.

The dendrogram exhibited 4 clear clusters. According to A perusal of the data given in Table 2 revealed that dendrogram at similarity index value 0.04 Jugahan-T was 3 all ten primers used were found to be polymorphic and separated from the rest of all progenies giving amplified the genomic DNA of 23 progenies of Ulmus information about its most diverse nature, which is villosa successfully. From the results of RAPD profiling categorized as cluster I. The remaining progenies number of RAPD markers generated per primer varied exhibited in 3 clear clusters. Overall cluster I was found to from 3 to 8 because of primer sequence and due to be 4 per cent similar with cluster II, III and IV. Cluster II is individual progeny (Table 2). All 10 primers produced the major cluster having 12 progenies viz., Jhidi - T , 3 distinct banding pattern for all the 23 progenies. 57 Jugahan-T and T , Jadh-T , T and T , Jugahan-T amplified products were detected and all were found to 1 5 1 4 2 4,

KatouchT , T and T , Jugahan-T and Jadh-T , which be polymorphic. Such a high level of polymorphism 2 3 5 2 3

exhibited 31 and 43 per cent similarity with cluster III and reflects the outcrossing nature of the species. Presently IV. Cluster III comprises of 6 progenies viz., Katouch-T , there seem to be no such studies conducted in U. villosa. 1

However, these findings receive support from high Jagoti- T , T , T , T and T , having 43 per cent similarity 1 2 3 4 5

frequencies of polymorphism of 78.30% in Melientha with cluster IV Cluster IV comprised of 4 progenies viz. .

suavis (Prathepha, 2000). Fourteen random RAPD primers amplified 118 loci, average of each primers is 8.4, 83 fragments (at a proportion of 70.34%) were polymorphic at the individual level in Ulmus pumila (Li et al., 2008). In Pterocarpus angolensis 75.3% polymorphism was detected (Amri and Mamboya, 2012). Similarly, Pharmawati et al. (2004) reported a high level of polymorphism, 99.39% for RAPD and 99.51% for ISSR, between Grevillea species. Twenty-eight per cent of genetic variations were found within populations in Melientha suavis (Prathepha, 2000). Goodall-Copestake et al. (2005) studied molecular markers and ex-situ conservation of the European elms (Ulmus spp.) using the 5 RAPD and 3 ISSR markers which yielded a total of 102 distinguishable bands and out of these 60 scored bands gave clear, reproducible, polymorphic characters which were scored as present or absent. Similarly Claros et al. (2000) observed 60 markers in olive-tree (Olea europaea L.) and 62 markers in Ficus polymorphic loci with RAPD markers to differentiate the varieties (Hadia et al., 2008). These findings also find support from Ma et al. (2012) in Elymus sibiricus where they detected 291 RAPD polymorphic loci in 93 samples. The percentage of polymorphic bands was 79%.

Jaccard's similarly correlation coefficient value ranged from 0.00 to 0.70. This suggested a fair range of variability in the similarity coefficient values indicating a broad genetic base of Ulmus villosa progenies. The highest value (0.70) was observed between Jag-T and 3

Jag-T which shows that they almost have the same 2

genetic constituents. Lowest similarity (0.00) was exhibited between some progenies. It may be attributed to the fact that all of those belong to different areas with different origin. Earlier studies by Weiguo and Yile (2004) while studying the genetic diversity in genus Morus using

Table 4: Banding pattern of 10 RAPD markers in 23 progenies of Ulmus villosa

JhT 2 JhT 3 JhT 5 JuT 1 JuT 2 JuT 3 JuT 4 JuT 5 JaT 1 JaT 2 JaT 3 JaT 4 JaT5 KaT 1 KaT 2 KaT 3 KaT 4 KaT 5 JagT 1 JagT 2 JagT 3 JagT4 JagT 5

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P A P A P A P A P A P A P

OPA-01

6

0

6

6

6

0

6

6

6

0

6

0

6

6

6

6

6

6

6

6

6

1

6

6

6

0

6

3

6

3

6

3

6

0 6 3 6 0 6 3 6 3 6 0 6 3

OPA-

04

3

0

3

0

3

0

3

3

3

0

3

1

3

3

3

3

3

3

3

3

3

0

3

0

3

3

3

1

3

2

3

2

3

0 3 3 3 3 3 2 3 1 3 2 3 3

0PA-

05

5

0

5

3

5

0

5

5

5

0

5

0

5

2

5

0

5

1

5

0

5

0

5

0

5

0

5

2

5

3

5

0

5

0 5 3 5 2 5 3 5 3 5 4 5 4

OPA-

07

8

0

8

1

8

0

8

5

8

1

8

0

8

1

8

2

8

0

8

2

8

0

8

0

8

0

8

1

8

4

8

3

8

0 8 4 8 2 8 2 8 1 8 3 8 1

OPA-

09

6

0

6

2

0

0

6

4

6

2

6

0

6

4

6

4

6

0

6

2

6

3

6

2

6

0

6

2

6

0

6

0

6

1 6 2 6 1 6 0 6 2 6 2 6 2 OPC-

08

7

0

7

1

7

0

7

0

7

1

7

0

7

3

7

2

7

0

7

0

7

1

7

1

7

0

7

0

7

0

7

1

7

0 7 2 7 2 7 2 7 2 7 2 7 0

OPC-

11

4

0

4

2

4

0

4

2

4

2

4

0

4

1

4

4

4

3

4

3

4

2

4

3

4

0

4

0

4

2

4

2

4

0 4 1 4 1 4 1 4 1 4 0 4 1

OPB-11 6 0 6 5 6 0 6 5 6 2 6 0 6 0 6 6 6 5 6 4 6 4 6 4 6 0 6 0 6 6 6 5 6 0 6 3 6 1 6 1 6 1 6 1 6 1 OPL- 06 6 4 6 0 6 3 6 5 6 0 6 0 6 6 6 6 6 5 6 5 6 0 6 5 6 5 6 0 6 5 6 0 6 4 6 0 6 1 6 2 6 2 6 2 6 0

OPS- 15 6 0 6 5 6 6 5 6 4 6 0 6 0 6 6 6 5 6 3 6 4 6 5 6 0 6 0 6 6 6 4 6 0 6 3 6 1 6 1 6 1 6 1 6 1

Total 4 25 3 40 12 1 26 39 28 28 15 26 8 9 31 20 5 24 14 17 17 17 16

A= Amplified P= Polymorphic

Table 5: Informative RAPD markers specific for a particular progeny

Primer Approximate size of DNA band Progeny

OPA-05 708.20 bp Jugahan, tree number 1

(Ju-T1) OPA-07 156.91bp

JhT1

JaT5

JhT5

KaT4

JhT3

JuT1

JuT5

JaT1

JaT4

JaT2

JuT4

KaT2

KaT3

KaT5

JuT2

JaT3

KaT1

JagT2

JagT3

JagT1

JagT4

JuT3

0.04 0.20 0.37 0.53 0.70

Coefficient

4

2

3

1

Fig. 1: Dendrogram based on UPGMA analysis 23 progenies of Ulmus villosa using RAPD markers

Fig. 2: RAPD fingerprints of 23 progenies revealed by OPA-09

Fig. 3: RAPD fingerprints of 23 progenies revealed by OPB-11

Fig. 4: RAPD fingerprints of 23 progenies revealed by OPS-15

Fig. 5: RAPD fingerprints of 23 progenies revealed by OPA-05

Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12252014]1224 The Indian Forester [December

observed between Jag-T and Jag-T . However, the Jhidi-T , Jadh-T , Jhidi-T and Katouch-T and showing 43 2 3 1 5 5 4

minimum similarity coefficient values (0.00) were per cent similarity with the rest of the progenies.observed in few progenies. The inference of Table 5 Discussion shows the informative RAPD markers specific for a

Molecular techniques have been found to be more particular progeny. Primer OPA-05 and OPA-07 produced

useful and accurate for determination of both unique bands of size approximately 708.20 bp and

interspecific and intraspecific genetic variation in plants. 156.91 bp for the JuT (Jugahan), respectively. These 1 Randomly amplified polymorphic DNA (RAPD) markers, informative primers producing unique bands for a

in particular, have been successfully employed for particular progeny can be specifically used for the study

determination of intraspecies genetic diversity in several of particular genotype.

plants. RAPD analysis has been successfully used for Cluster analysis differentiating species of a genus based on their

similarities and geographical proximities (Thomas et al., Dendrogram was created using the similarity 2001). In the present investigation RAPD markers used coefficient and un-weighted pair group method with for assay in genetic variation at molecular level showed arithmetic average (UPGMA) (Fig. 1). A critical perusal of that RAPDs were informative for revealing relationship dendrogram reveals that the distribution of various based upon similarity with in reference set of progenies.progenies into clusters and within cluster was random.

The dendrogram exhibited 4 clear clusters. According to A perusal of the data given in Table 2 revealed that dendrogram at similarity index value 0.04 Jugahan-T was 3 all ten primers used were found to be polymorphic and separated from the rest of all progenies giving amplified the genomic DNA of 23 progenies of Ulmus information about its most diverse nature, which is villosa successfully. From the results of RAPD profiling categorized as cluster I. The remaining progenies number of RAPD markers generated per primer varied exhibited in 3 clear clusters. Overall cluster I was found to from 3 to 8 because of primer sequence and due to be 4 per cent similar with cluster II, III and IV. Cluster II is individual progeny (Table 2). All 10 primers produced the major cluster having 12 progenies viz., Jhidi - T , 3 distinct banding pattern for all the 23 progenies. 57 Jugahan-T and T , Jadh-T , T and T , Jugahan-T amplified products were detected and all were found to 1 5 1 4 2 4,

KatouchT , T and T , Jugahan-T and Jadh-T , which be polymorphic. Such a high level of polymorphism 2 3 5 2 3

exhibited 31 and 43 per cent similarity with cluster III and reflects the outcrossing nature of the species. Presently IV. Cluster III comprises of 6 progenies viz., Katouch-T , there seem to be no such studies conducted in U. villosa. 1

However, these findings receive support from high Jagoti- T , T , T , T and T , having 43 per cent similarity 1 2 3 4 5

frequencies of polymorphism of 78.30% in Melientha with cluster IV Cluster IV comprised of 4 progenies viz. .

suavis (Prathepha, 2000). Fourteen random RAPD primers amplified 118 loci, average of each primers is 8.4, 83 fragments (at a proportion of 70.34%) were polymorphic at the individual level in Ulmus pumila (Li et al., 2008). In Pterocarpus angolensis 75.3% polymorphism was detected (Amri and Mamboya, 2012). Similarly, Pharmawati et al. (2004) reported a high level of polymorphism, 99.39% for RAPD and 99.51% for ISSR, between Grevillea species. Twenty-eight per cent of genetic variations were found within populations in Melientha suavis (Prathepha, 2000). Goodall-Copestake et al. (2005) studied molecular markers and ex-situ conservation of the European elms (Ulmus spp.) using the 5 RAPD and 3 ISSR markers which yielded a total of 102 distinguishable bands and out of these 60 scored bands gave clear, reproducible, polymorphic characters which were scored as present or absent. Similarly Claros et al. (2000) observed 60 markers in olive-tree (Olea europaea L.) and 62 markers in Ficus polymorphic loci with RAPD markers to differentiate the varieties (Hadia et al., 2008). These findings also find support from Ma et al. (2012) in Elymus sibiricus where they detected 291 RAPD polymorphic loci in 93 samples. The percentage of polymorphic bands was 79%.

Jaccard's similarly correlation coefficient value ranged from 0.00 to 0.70. This suggested a fair range of variability in the similarity coefficient values indicating a broad genetic base of Ulmus villosa progenies. The highest value (0.70) was observed between Jag-T and 3

Jag-T which shows that they almost have the same 2

genetic constituents. Lowest similarity (0.00) was exhibited between some progenies. It may be attributed to the fact that all of those belong to different areas with different origin. Earlier studies by Weiguo and Yile (2004) while studying the genetic diversity in genus Morus using

Table 4: Banding pattern of 10 RAPD markers in 23 progenies of Ulmus villosa

JhT 2 JhT 3 JhT 5 JuT 1 JuT 2 JuT 3 JuT 4 JuT 5 JaT 1 JaT 2 JaT 3 JaT 4 JaT5 KaT 1 KaT 2 KaT 3 KaT 4 KaT 5 JagT 1 JagT 2 JagT 3 JagT4 JagT 5

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P

A

P A P A P A P A P A P A P

OPA-01

6

0

6

6

6

0

6

6

6

0

6

0

6

6

6

6

6

6

6

6

6

1

6

6

6

0

6

3

6

3

6

3

6

0 6 3 6 0 6 3 6 3 6 0 6 3

OPA-

04

3

0

3

0

3

0

3

3

3

0

3

1

3

3

3

3

3

3

3

3

3

0

3

0

3

3

3

1

3

2

3

2

3

0 3 3 3 3 3 2 3 1 3 2 3 3

0PA-

05

5

0

5

3

5

0

5

5

5

0

5

0

5

2

5

0

5

1

5

0

5

0

5

0

5

0

5

2

5

3

5

0

5

0 5 3 5 2 5 3 5 3 5 4 5 4

OPA-

07

8

0

8

1

8

0

8

5

8

1

8

0

8

1

8

2

8

0

8

2

8

0

8

0

8

0

8

1

8

4

8

3

8

0 8 4 8 2 8 2 8 1 8 3 8 1

OPA-

09

6

0

6

2

0

0

6

4

6

2

6

0

6

4

6

4

6

0

6

2

6

3

6

2

6

0

6

2

6

0

6

0

6

1 6 2 6 1 6 0 6 2 6 2 6 2 OPC-

08

7

0

7

1

7

0

7

0

7

1

7

0

7

3

7

2

7

0

7

0

7

1

7

1

7

0

7

0

7

0

7

1

7

0 7 2 7 2 7 2 7 2 7 2 7 0

OPC-

11

4

0

4

2

4

0

4

2

4

2

4

0

4

1

4

4

4

3

4

3

4

2

4

3

4

0

4

0

4

2

4

2

4

0 4 1 4 1 4 1 4 1 4 0 4 1

OPB-11 6 0 6 5 6 0 6 5 6 2 6 0 6 0 6 6 6 5 6 4 6 4 6 4 6 0 6 0 6 6 6 5 6 0 6 3 6 1 6 1 6 1 6 1 6 1 OPL- 06 6 4 6 0 6 3 6 5 6 0 6 0 6 6 6 6 6 5 6 5 6 0 6 5 6 5 6 0 6 5 6 0 6 4 6 0 6 1 6 2 6 2 6 2 6 0

OPS- 15 6 0 6 5 6 6 5 6 4 6 0 6 0 6 6 6 5 6 3 6 4 6 5 6 0 6 0 6 6 6 4 6 0 6 3 6 1 6 1 6 1 6 1 6 1

Total 4 25 3 40 12 1 26 39 28 28 15 26 8 9 31 20 5 24 14 17 17 17 16

A= Amplified P= Polymorphic

Table 5: Informative RAPD markers specific for a particular progeny

Primer Approximate size of DNA band Progeny

OPA-05 708.20 bp Jugahan, tree number 1

(Ju-T1) OPA-07 156.91bp

JhT1

JaT5

JhT5

KaT4

JhT3

JuT1

JuT5

JaT1

JaT4

JaT2

JuT4

KaT2

KaT3

KaT5

JuT2

JaT3

KaT1

JagT2

JagT3

JagT1

JagT4

JuT3

0.04 0.20 0.37 0.53 0.70

Coefficient

4

2

3

1

Fig. 1: Dendrogram based on UPGMA analysis 23 progenies of Ulmus villosa using RAPD markers

Fig. 2: RAPD fingerprints of 23 progenies revealed by OPA-09

Fig. 3: RAPD fingerprints of 23 progenies revealed by OPB-11

Fig. 4: RAPD fingerprints of 23 progenies revealed by OPS-15

Fig. 5: RAPD fingerprints of 23 progenies revealed by OPA-05

1226 The Indian Forester [December

Acknowledgments

The authors are thankful to the Professor and Head, Department of Tree Improvement and Genetic Resources for

providing all the necessary facilities to carry out the present studies. The help rendered by the State Department of Forest,

Himachal Pradesh is also duly acknowledged.

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References

Alstrom Rapaport C., Lascoux M., Wang Y.C., Roberts G. and Tuskan G.A. (1998). Identification of a RAPD Marker Linked to Sex Determination

in the Basket Willow (Salix viminalis L.). Journal of Heredity, 89:44–49.

Amri E. and Mamboya F. (2012). Genetic diversity in Pterocarpus angolensis populations detected by Random Amplified Polymorphic DNA

markers. International Journal of Plant Breeding and Genetics, 6(2):105-114.

Anamthawat Jonsson. K. Thorsson A.T. (2003) Natural hybridization in birch: triploid hybrids between Betula nana and B. pubescens.

Plant Cell Tissue Organ Cult., 75:99–107.

and

RAPD markers, revealed the highest similarity (0.9912) closely related to the north American species than to the between T and T (Thailand). The result indicated that south American P. pallida or P. chilensis. 11 12

they almost have the same genetic constituents. The The dendrogram exhibited four clear clusters and least similarity between Broussonetia papyrifera and according to it Jugahan-T (at similarity index value 0.04) 3

Yaan 3 was ascribed to their genome difference because was separated from the rest of all progenies giving B. papyrifera and Yaan 3 were from different genus based information about its most diverse nature, which is on the molecular Jaccard matrix in molecular categorized as cluster I. The remaining progenies characterization of mulberry germplasm using RAPD exhibited in 3 clear clusters. Overall cluster I was found to primers. Similarly Ozrenk et al. (2010) reported that the be 4 per cent similar with cluster II, III and IV. Cluster II is most similar genotypes (0.93) were D5 (a genotype from the major cluster having 12 progenies viz., Jhidi - T , 3

Erzincan) and DE5 (a genotype from Elazig) followed by Jugahan-T and T , Jadh-T , T and T , Jugahan-T1 5 1 4 2 4,

D6 and D7 (two genotypes from Erzincan) (0.90) and they KatouchT , T and T , Jugahan-T and Jadh-T , which 2 3 5 2 3concluded that the genetic similarities among the

exhibited 31 and 43 per cent similarity with cluster III and genotypes grown in the same region were generally

IV. Cluster III comprises of 6 progenies viz., Katouch-T , 1found close because they had been reproduced from the

Jagoti- T , T , T , T and T , having 43 per cent similarity 1 2 3 4 5 similar genotypes. On the other hand different adapted with cluster IV Cluster IV comprised of 4 progenies viz. . genotypes from the same region could be the Jhidi-T , Jadh-T , Jhidi-T and Katouch-T and showing 43 1 5 5 4introductions from various other regions. Jaccard per cent similarity with the rest of the progenies.similarity coefficient ranged from 0.66 to 0.95 showing a

These results are in conformity with the findings of wide range of variability among the clones of Dalbergia Nanda et al. (2004). They also reported similar studies in sissoo as analysed by RAPD markers. One more similar Acacia. Genetic similarity matrix coefficient indicated study was carried out by Sherry et al. (2011) in Prosopis that Acacia catechu had about 11%, 29% and 31% spp. and reported that highest similarity was shown similarity with A. mollissima, A. arabica and A. between the north American species, P. articulata, P. farensiana, respectively. The cluster analysis indicated velutina, P. glandulosa and P. laevigata with an index of that six species of Acacia formed two major clusters. The 0.67 between them. Similarly, the index between all the first major cluster represented by only one species each north American species selected in the study was i.e. A. arabica. Second major cluster was represented by relatively high showing a strong relation in terms of five species i.e. A. catechu, A. farnesiana, A. geographical proximity. The distance between P. pallida auriculiformis, A. concinna and A. mollissima. A. and P. julifora was only 0.27 compared with 0.46 between farnesiana and A. catechu representing a minor cluster P. juliflora and P. velutina. P. julifora appeared to be more

Table 6: Similarity coefficient values of RAPD data using Jaccard's Similarity correlation coefficient

Site JhT2 JhT 3 JhT 5 JuT 1 JuT 2 JuT 3 JuT 4 JuT 5 JaT 1 JaT 2 JaT 3 JaT 4 JaT 5 KaT 1 KaT 2 KaT 3 KaT4 KaT 5 JagT 1 JagT 2 JagT 3 JagT4 JagT 5

JhT2 1.00 JhT3

0.00

1.00

JhT5

0.40

0.00

1.00

JuT1

0.10

0.54

0.07

1.00

JuT2

0.00

0.23

0.00

0.18

1.00

JuT3

0.00

0.00

0.00

0.02

0.00

1.00

JuT4

0.15

0.34

0.11

0.50

0.11

0.03

1.00

JuT5

0.10

0.52

0.07

0.68

0.27

0.02

0.54

1.00

JaT1

0.14

0.47

0.10

0.58

0.25

0.03

0.42

0.67

1.00

JaT2

0.14

0.33

0.11

0.52

0.30

0.03

0.47

0.65

0.66

1.00

JaT3

0.00

0.29

0.00

0.25

0.42

0.00

0.13

0.31

0.34

0.35

1.00

JaT4

0.15

0.45

0.11

0.46

0.26

0.00

0.36

0.58

0.68

0.65

0.51

1.00

JaT5

0.50

0.00

0.37

0.20

0.00

0.12

0.30

0.20

0.28

0.29

0.00

0.17

1.00

KaT1

0.00

0.25

0.00

0.19

0.05

0.11

0.29

0.14

0.15

0.16

0.09

0.12

0.06

1.00

KaT2

0.09

0.47

0.09

0.57

0.26

0.03

0.32

0.55

0.59

0.45

0.31

0.46

0.18

0.21

1.00

KaT3

0.00

0.36

0.00

0.36

0.33

0.05

0.17

0.40

0.41

0.34

0.29

0.31

0.07

0.20

0.59

1.00

KaT4

0.50

0.03

0.60

0.12

0.00

0.00

0.19

0.12

0.13

0.14

0.00

0.14

0.44

0.07

0.12

0.00

1.00

KaT5

0.00

O.41

0.00

0.43

0.22

0.03

0.33

0.38

0.38

0.39

0.24

0.30

0.09

0.29

0.54

0.48

0.03

1.00

JagT1

0.05

0.14

0.00

0.22

0.13

0.07

0.29

0.17

0.23

0.20

0.16

0.14

0.22

0.21

0.28

0.25

0.05

0.37

1.00

JagT2 0.10 0.27 0.00 0.29 0.11 0.05 0.34 0.21 0.32 0.29 0.18 0.26 0.19 0.36 0.41 0.37 0.04 0.43 0.63 1.00

JagT3 0.10 0.31 0.00 0.32 0.11 0.05 0.38 0.24 0.28 0.25 0.18 0.26 0.13 0.44 0.33 0.27 0.10 0.43 0.47 0.70 1.00

JagT4 0.10 0.20 0.00 0.29 0.11 0.05 0.30 0.19 0.18 0.18 0.10 0.13 0.19 0.23 0.29 0.19 0.10 0.38 0.63 0.61 0.54 1.00JagT5 0.00 0.32 0.00 0.36 0.12 0.06 0.35 0.25 0.29 0.26 0.14 0.16 0.14 0.38 0.30 0.28 0.05 0.50 0.42 0.50 0.57 0.43 1.00

with about 30% similarity. A. auriculiformis shared about clusters. The sub-cluster II consisted maximum of twelve 28% similarity with A. farnesiana and A. catechu. A. clones followed by cluster I (11 clones) and cluster VI (7 mollissima shared about 18% similarity with A. arabica. clones) whereas clusters IV, VII and VIII consisted single One minor cluster comprises A. farensiana and A. clone each. Ambri and Mamboya (2012) got three major catechu which were about 30% similarity because of the clusters while studying within and between population close association with regards to growth habit and diversity of Pterocarpus amgolensis in Tanzania. They taxonomical classification. observed high genetic variations within populations

(77.13%) and low among populations (22.86%). Normally dispersal of seeds which makes partitioning and distribution of variability complex has Conclusionbeen cited as reason for the grouping of genotypes to one Molecular techniques have been found to be more cluster collected from different places. It may be true in useful and accurate for determination of both this study also. study did not show the separation of interspecific and intraspecific genetic variation in plants. individuals from different agro-climatic sub-zones of the Randomly amplified polymorphic DNA (RAPD) markers, same geographic origin. Conversely, in several other in particular, have been successfully employed for studies which involved various species/accessions per determination of intraspecies genetic diversity in several geographic origin, genetic variations were observed to be plants. Besides this, the RAPD technology has received a conserved with geographic origin as revealed by Josiah et great deal of attention for genetic diversity studies al. (2008) in Acacia senegal. studied the genetic diversity because of its simplicity and rapidity. The RAPD profile using RAPD markers in Butea monosperma and clustered usually represent available portion of the genome. The genotypes into three groups. The cluster one was present result thus highlights the utility of RAPD marker represented by seven samples. All samples of this group in providing great information on the genetic structure of belonged to semi-arid agro-ecological major zone and U. villosa progenies. Markers were also helpful in three agro-climatic sub- zones. Similarly Kumar et al., characterizing U. villosa progenies. The clustering (2011) in the assessment of genetic diversity in different pattern exhibited that the geographic distribution Dalbergia sissoo clones by RAPD markers grouped clones provides true index of genetic diversity in progenies of U. into two clusters which were further divided into sub- villosa.

Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12272014]

1226 The Indian Forester [December

Acknowledgments

The authors are thankful to the Professor and Head, Department of Tree Improvement and Genetic Resources for

providing all the necessary facilities to carry out the present studies. The help rendered by the State Department of Forest,

Himachal Pradesh is also duly acknowledged.

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References

Alstrom Rapaport C., Lascoux M., Wang Y.C., Roberts G. and Tuskan G.A. (1998). Identification of a RAPD Marker Linked to Sex Determination

in the Basket Willow (Salix viminalis L.). Journal of Heredity, 89:44–49.

Amri E. and Mamboya F. (2012). Genetic diversity in Pterocarpus angolensis populations detected by Random Amplified Polymorphic DNA

markers. International Journal of Plant Breeding and Genetics, 6(2):105-114.

Anamthawat Jonsson. K. Thorsson A.T. (2003) Natural hybridization in birch: triploid hybrids between Betula nana and B. pubescens.

Plant Cell Tissue Organ Cult., 75:99–107.

and

RAPD markers, revealed the highest similarity (0.9912) closely related to the north American species than to the between T and T (Thailand). The result indicated that south American P. pallida or P. chilensis. 11 12

they almost have the same genetic constituents. The The dendrogram exhibited four clear clusters and least similarity between Broussonetia papyrifera and according to it Jugahan-T (at similarity index value 0.04) 3

Yaan 3 was ascribed to their genome difference because was separated from the rest of all progenies giving B. papyrifera and Yaan 3 were from different genus based information about its most diverse nature, which is on the molecular Jaccard matrix in molecular categorized as cluster I. The remaining progenies characterization of mulberry germplasm using RAPD exhibited in 3 clear clusters. Overall cluster I was found to primers. Similarly Ozrenk et al. (2010) reported that the be 4 per cent similar with cluster II, III and IV. Cluster II is most similar genotypes (0.93) were D5 (a genotype from the major cluster having 12 progenies viz., Jhidi - T , 3

Erzincan) and DE5 (a genotype from Elazig) followed by Jugahan-T and T , Jadh-T , T and T , Jugahan-T1 5 1 4 2 4,

D6 and D7 (two genotypes from Erzincan) (0.90) and they KatouchT , T and T , Jugahan-T and Jadh-T , which 2 3 5 2 3concluded that the genetic similarities among the

exhibited 31 and 43 per cent similarity with cluster III and genotypes grown in the same region were generally

IV. Cluster III comprises of 6 progenies viz., Katouch-T , 1found close because they had been reproduced from the

Jagoti- T , T , T , T and T , having 43 per cent similarity 1 2 3 4 5 similar genotypes. On the other hand different adapted with cluster IV Cluster IV comprised of 4 progenies viz. . genotypes from the same region could be the Jhidi-T , Jadh-T , Jhidi-T and Katouch-T and showing 43 1 5 5 4introductions from various other regions. Jaccard per cent similarity with the rest of the progenies.similarity coefficient ranged from 0.66 to 0.95 showing a

These results are in conformity with the findings of wide range of variability among the clones of Dalbergia Nanda et al. (2004). They also reported similar studies in sissoo as analysed by RAPD markers. One more similar Acacia. Genetic similarity matrix coefficient indicated study was carried out by Sherry et al. (2011) in Prosopis that Acacia catechu had about 11%, 29% and 31% spp. and reported that highest similarity was shown similarity with A. mollissima, A. arabica and A. between the north American species, P. articulata, P. farensiana, respectively. The cluster analysis indicated velutina, P. glandulosa and P. laevigata with an index of that six species of Acacia formed two major clusters. The 0.67 between them. Similarly, the index between all the first major cluster represented by only one species each north American species selected in the study was i.e. A. arabica. Second major cluster was represented by relatively high showing a strong relation in terms of five species i.e. A. catechu, A. farnesiana, A. geographical proximity. The distance between P. pallida auriculiformis, A. concinna and A. mollissima. A. and P. julifora was only 0.27 compared with 0.46 between farnesiana and A. catechu representing a minor cluster P. juliflora and P. velutina. P. julifora appeared to be more

Table 6: Similarity coefficient values of RAPD data using Jaccard's Similarity correlation coefficient

Site JhT2 JhT 3 JhT 5 JuT 1 JuT 2 JuT 3 JuT 4 JuT 5 JaT 1 JaT 2 JaT 3 JaT 4 JaT 5 KaT 1 KaT 2 KaT 3 KaT4 KaT 5 JagT 1 JagT 2 JagT 3 JagT4 JagT 5

JhT2 1.00 JhT3

0.00

1.00

JhT5

0.40

0.00

1.00

JuT1

0.10

0.54

0.07

1.00

JuT2

0.00

0.23

0.00

0.18

1.00

JuT3

0.00

0.00

0.00

0.02

0.00

1.00

JuT4

0.15

0.34

0.11

0.50

0.11

0.03

1.00

JuT5

0.10

0.52

0.07

0.68

0.27

0.02

0.54

1.00

JaT1

0.14

0.47

0.10

0.58

0.25

0.03

0.42

0.67

1.00

JaT2

0.14

0.33

0.11

0.52

0.30

0.03

0.47

0.65

0.66

1.00

JaT3

0.00

0.29

0.00

0.25

0.42

0.00

0.13

0.31

0.34

0.35

1.00

JaT4

0.15

0.45

0.11

0.46

0.26

0.00

0.36

0.58

0.68

0.65

0.51

1.00

JaT5

0.50

0.00

0.37

0.20

0.00

0.12

0.30

0.20

0.28

0.29

0.00

0.17

1.00

KaT1

0.00

0.25

0.00

0.19

0.05

0.11

0.29

0.14

0.15

0.16

0.09

0.12

0.06

1.00

KaT2

0.09

0.47

0.09

0.57

0.26

0.03

0.32

0.55

0.59

0.45

0.31

0.46

0.18

0.21

1.00

KaT3

0.00

0.36

0.00

0.36

0.33

0.05

0.17

0.40

0.41

0.34

0.29

0.31

0.07

0.20

0.59

1.00

KaT4

0.50

0.03

0.60

0.12

0.00

0.00

0.19

0.12

0.13

0.14

0.00

0.14

0.44

0.07

0.12

0.00

1.00

KaT5

0.00

O.41

0.00

0.43

0.22

0.03

0.33

0.38

0.38

0.39

0.24

0.30

0.09

0.29

0.54

0.48

0.03

1.00

JagT1

0.05

0.14

0.00

0.22

0.13

0.07

0.29

0.17

0.23

0.20

0.16

0.14

0.22

0.21

0.28

0.25

0.05

0.37

1.00

JagT2 0.10 0.27 0.00 0.29 0.11 0.05 0.34 0.21 0.32 0.29 0.18 0.26 0.19 0.36 0.41 0.37 0.04 0.43 0.63 1.00

JagT3 0.10 0.31 0.00 0.32 0.11 0.05 0.38 0.24 0.28 0.25 0.18 0.26 0.13 0.44 0.33 0.27 0.10 0.43 0.47 0.70 1.00

JagT4 0.10 0.20 0.00 0.29 0.11 0.05 0.30 0.19 0.18 0.18 0.10 0.13 0.19 0.23 0.29 0.19 0.10 0.38 0.63 0.61 0.54 1.00JagT5 0.00 0.32 0.00 0.36 0.12 0.06 0.35 0.25 0.29 0.26 0.14 0.16 0.14 0.38 0.30 0.28 0.05 0.50 0.42 0.50 0.57 0.43 1.00

with about 30% similarity. A. auriculiformis shared about clusters. The sub-cluster II consisted maximum of twelve 28% similarity with A. farnesiana and A. catechu. A. clones followed by cluster I (11 clones) and cluster VI (7 mollissima shared about 18% similarity with A. arabica. clones) whereas clusters IV, VII and VIII consisted single One minor cluster comprises A. farensiana and A. clone each. Ambri and Mamboya (2012) got three major catechu which were about 30% similarity because of the clusters while studying within and between population close association with regards to growth habit and diversity of Pterocarpus amgolensis in Tanzania. They taxonomical classification. observed high genetic variations within populations

(77.13%) and low among populations (22.86%). Normally dispersal of seeds which makes partitioning and distribution of variability complex has Conclusionbeen cited as reason for the grouping of genotypes to one Molecular techniques have been found to be more cluster collected from different places. It may be true in useful and accurate for determination of both this study also. study did not show the separation of interspecific and intraspecific genetic variation in plants. individuals from different agro-climatic sub-zones of the Randomly amplified polymorphic DNA (RAPD) markers, same geographic origin. Conversely, in several other in particular, have been successfully employed for studies which involved various species/accessions per determination of intraspecies genetic diversity in several geographic origin, genetic variations were observed to be plants. Besides this, the RAPD technology has received a conserved with geographic origin as revealed by Josiah et great deal of attention for genetic diversity studies al. (2008) in Acacia senegal. studied the genetic diversity because of its simplicity and rapidity. The RAPD profile using RAPD markers in Butea monosperma and clustered usually represent available portion of the genome. The genotypes into three groups. The cluster one was present result thus highlights the utility of RAPD marker represented by seven samples. All samples of this group in providing great information on the genetic structure of belonged to semi-arid agro-ecological major zone and U. villosa progenies. Markers were also helpful in three agro-climatic sub- zones. Similarly Kumar et al., characterizing U. villosa progenies. The clustering (2011) in the assessment of genetic diversity in different pattern exhibited that the geographic distribution Dalbergia sissoo clones by RAPD markers grouped clones provides true index of genetic diversity in progenies of U. into two clusters which were further divided into sub- villosa.

Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12272014]

Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12292014]1228 The Indian Forester [December

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and

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Estimation of genetic diversity in progenies of selected genotypes of Ulmus villosa Brandis .... 12292014]1228 The Indian Forester [December

Rout G.R., Bhatacharya N.R.M., Nayak S. and Das P. (2003). Evaluation of genetic relationships in Dalbergia species using RAPD markers. Biodiversity and Conservation, 12: 197-206.

Santamour F.S., (1979). Resistance of Himalayan small-leaved elm to Dutch elm disease. Journal of Arboriculture, 5:110-112.

Sherry M., Smith S., Patel A., Harris P., Hand P., Trenchard L. and Henderson J., (2011). RAPD and microsatellite transferability studies in selected species of Prosopis (section Algarobia) with emphasis on Prosopis juliflora and P. pallid. Journal of Genetics, 90(2): 251-264.

Singh K.B., (1991). Study on the propagation techniques of Ulmus laevigata Royle (Vern. Marinoo) M.Sc. Thesis, Dr. Y.S. Parmar, UHF, Nauni, Solan (H.P). 2p.

Singh R.V., (1982). Fodder trees of India. Oxford and IBH Publishing Co. Ptv. Ltd. NewDelhi, India, 663p.

Skepner A.P. and Krane D.E. (1998). RAPD reveals genetic similarity of Acer saccharum and Acer nigrum. Heredity, 80:422–428.

Sun Y., Zhao Y., Wang X., Qiao G., Chen G., Yang Y., Zhou J., Jin L. and Zhou R. (2009). Adventitious bud regeneration from leaf explants of Platanus occidentalis L. and genetic stability assessment. Acta Physiol Plant., 31: 33–41.

Thomas G., Sreejayan J.L., and Kuriachen P., (2001). Genetic variation and population structure in Oryza malampuzhaensis endemic to Western Ghats, South. Indian Journal of Genetics, 80: 141-148.

Vigouroux A., Besnard G., Sossey-Alaoui K., Tersac M. and Berville A. (1997). Hybrid origin of Platanus acerifolia confirmed and that of P. densicoma revealed using molecular markers. Acta Bot. Gall., 144:243–252.

Weiguo Zhao and Yile Pan., (2004). Genetic diversity of genus Morus revealed by RAPD markers. Indian. Journal of Agricultural Biology, 6(6): 950-955.

Williams J.G.K., Kubelik A.R., Levak K.J., Rafaski J.A. and Tingey S.V. (1990). DNA polymorphism amplified by arbitrary primer are useful as genetic marker. Nucleic Acid Research, 18: 6531-6535.

www.iucnredlist.org: International Union for Conservation of Nature and Natural Resources.

Anderson W.R. and Fairbanks D.J. (1990). Molecular markers: Important tools for plant genetic resource characterization. Diversity, 6:

51–53.

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INDIAN MEDICINAL AND AROMATIC PLANTS - PART I AND II

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