123 1. Introduction Persian walnut (Juglans regia L.) is one of the most important nut crops grown in temperate regions and it produces edible nuts of high nutritional value. In In- dia there are no systematic orchards of walnut contain- ing standard cultivars. Although some efforts have been made in this direction in the last two decades and the superior selections from the seedling populations were selected through field surveys in different walnut grow- ing districts of Himachal Pradesh (Thakur, 1993; Gupta, 1999; Sharma, 1999, 2002; Thakur et al., 2005). Howev- er, a large number of exotic accessions have also been in- troduced from abroad and a germplasm collection block has been maintained after propagation of these selections and exotic accessions on the seedling rootstock. There- fore characterization and evaluation of this germplasm is important for future use and a wider divergence is a pre- requisite for breeding purposes. Information about the extent of genetic divergence is critical for the improve- ment of any crop in order to have heterotic responses and desirable segregants. Furthermore, information on the nature and degree of genetic divergence present in the collected germplasm could help for further improvement through hybridization. 2. Materials and Methods The present investigation was undertaken on three- to four-year-old plants for a total of 54 indigenous selec- tions collected through field survey at different loca- tions in Himachal Pradesh and exotic cultivars of Persian walnut introduced directly from abroad (Table 1). These were propagated on seedling rootstocks and are presently growing at Oachghat Block of Dr. Y.S. Parmar Univer- sity of Horticulture and Forestry, Nauni-Solan (Himachal Pradesh) located around 1275 m above mean sea level and between 31oN latitude and 77oE longitude. Data for various growth [plant height (m), trunk girth (cm2), plant spread (cm), TCSA (cm2) and plant volume (cm3)] and foliage characters [leaf length (cm), leaf width (cm), leaf area (cm2), leaflet length (cm), leaflet width (cm) and number of leaflets] were recorded. Ten compound leaves were taken from each individual tree for foliage characters and average means were calculated. Standard procedures were followed to calculate the mean, standard deviation and coefficient of variation for various char- acters as described by Panse and Sukhatme (1995). The genetic divergence among the accessions was estimated by Mahalanobis D2 statistic as suggested by Rao (1952). Genetic divergence studies regarding different growth and foliage characters of walnut (Juglans regia L.) germplasm S. Sharma*, K. Kumar** (1), A. Kumar*** * Division of Fruit Science, SKUAST, Jammu, Jammu and Kashmir, India. ** Department of Fruit Science, UHF, Nauni-Solan, Himachal Pradesh, India. *** Division of Fruit Science, SKUAST-Kashmir, Jammu and Kashmir, India. Key words: divergence, exotic cultivars, growth, local selections, walnut germplasm. Abstract: Genetic divergence of 29 exotic walnut cultivars introduced from abroad and 25 local selections from seedling population was studied. The analysis of variance revealed significant differences among accessions for each character un- der study. Based on Mahalanobis D2 values the accessions were grouped into eight clusters. Cluster I had a maximum of 12 accessions followed by cluster II with 11 accessions. The mean intra and inter cluster distance (D) revealed that cluster VII had the highest intra cluster distance (2.171), while the inter cluster distance was the greatest between cluster II and IV (10.528). Characters like plant height, trunk girth, plant spread, plant volume, leaf length, leaf width and number of leaflets contributed the most to total divergence. Adv. Hort. Sci., 2014 28(3): 123-128 (1) Corresponding author: khokherak@rediffmail.com Received for publication 15 August 2013 Accepted for publication 11 September 2014 124 All the accessions were grouped into clusters according to the Tocher’s method described in Rao (1952). 3. Results and Discussion The analysis of variance revealed significant differ- ences among the genotypes for each character, indicat- ing the existence of variability among the genotypes for such characters. On the basis of the relative magnitude of D2 values, 54 accessions were grouped, using Toch- er’s method, into eight clusters. Cluster I was the larg- est with 12 accessions followed by cluster II having 11 accessions, and cluster V having 10 accessions; clusters III, IV, VI, VII, VIII had 5, 1, 9, 4, 2 accessions, respec- tively (Fig. 1 and Table 2). The highest intra cluster dis- tance was registered in cluster VII (2.171) followed by cluster II (1.951), while the minimum intra cluster value was exhibited by cluster IV (0.000). The minimum in- ter-cluster D2 value was recorded between cluster I and V (1.821), indicating a close relationship and similar- ity of most traits of the accessions. Hence, selection of parents from these clusters should be avoided. However, the highest inter-cluster D2 value was observed between clusters II and IV (10.528), followed by clusters I and IV (10.411), confirming a wide genetic distance between these groups (Table 3). Since these clusters exhibited the greatest inter-cluster distances, selection of parents from such clusters for hybridization programmes could help to develop novel hybrids. A similar trend of clustering patterns has been reported by Barua and Sharma (2003) in apple, Thakur et al. (2005) in almond., Kaushal and Sharma (2005) in pecan, and Sharma and Sharma (2005), Pandey and Tripathi (2007), and Sharma et al. (2010) in walnut. The mean value, standard deviation and co-efficient of variability on various growth and foliage traits of clus- ter is presented in Table 4. Cluster IV had the highest mean values for characters plant height (5.50 m), trunk girth (33.00 cm) and TCSA (86.55 cm2), while cluster VIII exhibited the highest mean values for plant spread (110.00 cm), plant volume (1.71 cm3), leaf length (56.50 cm), leaf width (30.25 cm), leaf area (57.35 cm2), leaf- let length (16.17 cm). Maximum mean values for leaf- let width (7.19 cm) and number of leaflets (9.73) was recorded in clusters VI and II, respectively. Clusters IV and VIII can be expected to give promising and desirable recombinations in segregating generations because they comprise desirable features as revealed from their cluster means. Similarly, maximum co-efficient of variability for plant height (40.00%), trunk girth (33.11%), plant spread (42.08%), TCSA (65.21%), plant volume (87.50%) and number of leaflets (23.12%) was observed in cluster II, however, cluster I had the highest co-efficient of variabil- ity for traits leaf area (32.86%), leaflet length (13.46%) and leaflet width (19.79%). In the principal component analysis presented in Table 5, the first vector shows the highest eigen value (4.332) Table 1 - Different exotic and indigenous walnut accessions and their place of origin S. No. Accession Place of origin Exotic cultivars 1. Adams 10 USA 2. Aksu≠24 China 3. Aksu≠71 China 4. Aksu≠81 China 5. Aksu≠210 China 6. Aksu≠417 China 7. Aksu Hun ≠85 China 8. Bulgaria 3 Bulgaria 9. Chandler USA 10. Chico USA 11. Cisco USA 12. Conway Mayette USA 13. Corne France 14. Graves Franquette USA 15. Hotien 8 China 16. Howard USA 17. Howe USA 18. Mayette USA 19. Meylannaise France 20. Nn 88 Godyn Poland 21. Parisienne France 22. Placentia USA 23. Ronde de Montignac France 24. Scharsch Franquette USA 25. Serr USA 26. Shinrei China 27. Xin Zad Fen China 28. Xin Zheng Zhu China 29. Zhong Lin≠3 China Indigenous selections 30. Brij Lal Selection H.P. (India) 31. Chamba Selection-20 H.P. (India) 32. Chamba Selection-32 H.P. (India) 33. Chamba Selection-60 H.P. (India) 34. Chamba Selection-101 H.P. (India) 35. Chamba Selection-123 H.P. (India) 36. Daulat Ram Selection H.P. (India) 37. Jaunaji Selection -2 H.P. (India) 38. Jaunaji Selection –4 H.P. (India) 39. Jaunaji Selection –6 H.P. (India) 40. Jaunaji Selection-7 H.P. (India) 41. Jaunaji Selection-10 H.P. (India) 42. Jaunaji Selection-12 H.P. (India) 43. Jogindernagar Selection-23 H.P.(India) 44. Jogindernagar Selection-39 H.P. (India) 45. Jogindernagar Selection-61 H.P. (India) 46. Jogindernagar Selection-122 H.P. (India) 47. Nauradhar Selection-10 H.P. (India) 48. Nauradhar Selection-36 H.P. (India) 49. Nauradhar Selection-53 H.P. (India) 50. Rajgarh Selection-1 H.P. (India) 51. Rajgarh Selection-8 H.P. (India) 52. Rajgarh Selection-11 H.P. (India) 53. Selection No. 8 H.P. (India) 54. Selection No. 51 H.P. (India) 125 and accounts for 39.38% of the total variation. The first vector is the combination of leaf length, trunk girth, plant height and leaflet length. The second vector has an eigen value of 3.008 and explains 27.34% of total variation, and this factor is mainly the combination of number of leaflets and trunk girth. The third vector has an eigen value of 1.155 and total variation of 10.50%, from the combination of leaf width and plant spread. The fourth vector has an eigen value of 1.047 and total variation of 9.52%, with the maximum contribution of trunk girth. Vector fifth has an eigen value of 0.424 and total varia- tion of 3.85%, mainly a combination of plant volume and plant spread. The sixth vector has an eigen value of 0.347 with total variation of 3.15 % and the highest contribu- Table 2 - Distribution of 54 walnut accessions into various clusters Clusters Number of accessions Accessions I 12 Aksu≠210, Chamba Selection-32, Chamba Selection-60, Conway Mayette, Hotien 8, Jaunaji Selection-2, Jaunaji Selection-12, Jogindernagar Selection-61, Nauradhar Selection-10, Parisienne, Ronde de Montig- nac, Xin Zheng Zhu II 11 Adams 10, Aksu≠24, Chamba Selection-101, Jaunaji Selection-7, Jaunaji Selection-10, Jogindernagar Se- lection-39, Jogindernagar Selection-122, Meylannaise, Nauradhar Selection-36, Nauradhar Selection-53, Selection No. 8. III 5 Aksu≠81, Cisco, Daulat Ram Selection, Jaunaji Selection-4, Scharsch Franquette IV 1 Howard V 10 Aksu≠71, Aksu≠417, Bulgaria 3, Graves Franquette, Howe, Jaunaji Selection-6, Mayette, Rajgarh Selec- tion-1, Selection No. 51, Xin Zad Fen VI 9 Aksu Hun≠85, Brij Lal Selection, Chico, Corne, Jogindernagar Selection-23, Nn 88 Godyn, Serr, Shinrei, Zhong Lin ≠3 VII 4 Chamba Selection-20, Placentia, Rajgarh Selection-8, Rajgarh Selection-11 VIII 2 Chamba Selection-123, Chandler Fig. 1 - Dendrogram showing different clusters of genotypes, grouped by Tocher’s method, in walnut accessions. 126 Table 4 - Mean, standard deviation and coefficient of variation for various clusters on the basis of growth and foliage characters of walnut germplasm S. No. Characters Parameters Clusters I II III IV V VI VII VIII 1 Plant height (m) Mean 1.12 1.00 2.98 5.50 1.36 1.77 1.91 2.65 SD 0.27 0.40 0.85 0.00 0.52 0.49 0.61 0.21 COV 24.11 40.00 28.52 0.00 38.23 27.68 31.94 7.92 2 Trunk girth (cm) Mean 7.15 7.55 10.40 33.00 9.45 10.75 12.12 16.00 SD 2.05 2.50 2.25 0.00 2.60 1.70 3.12 2.83 COV 28.67 33.11 21.63 0.00 27.51 15.81 25.74 17.69 3 Plant spread (cm) Mean 31.71 36.36 44.00 50.00 39.50 51.94 70.00 110.00 SD 11.48 15.30 13.87 0.00 12.95 17.36 17.80 14.14 COV 36.20 42.08 31.52 0.00 32.78 33.42 25.43 12.85 4 TCSA (cm2) Mean 4.38 5.03 8.91 86.55 7.61 8.27 12.28 20.74 SD 2.17 3.28 3.29 0.00 4.56 2.99 6.10 7.25 COV 49.54 65.21 36.92 0.00 59.92 36.15 49.67 34.96 5 Plant volume (cm3) Mean 0.07 0.08 0.32 0.72 0.15 0.30 1.57 1.71 SD 0.06 0.07 0.21 0.00 0.11 0.25 0.76 0.57 COV 85.71 87.50 65.62 0.00 73.33 83.33 48.41 33.33 6 Leaf length (cm) Mean 33.46 46.73 35.00 40.00 27.05 39.33 32.62 56.50 SD 5.47 3.98 2.89 0.00 3.46 4.84 6.94 6.36 COV 16.35 8.52 8.26 0.00 12.79 12.31 21.27 11.26 7 Leaf width(cm) Mean 18.55 24.97 18.95 20.50 15.50 22.58 18.06 30.25 SD 2.90 2.26 2.15 0.00 2.41 3.97 1.88 0.35 COV 15.63 9.05 11.34 0.00 15.55 17.58 10.41 1.16 8 Leaf area (cm2) Mean 29.73 56.69 33.34 38.39 26.28 48.00 38.10 57.35 SD 9.77 8.90 8.12 0.00 6.25 8.64 9.60 2.96 COV 32.86 15.70 24.35 0.00 23.78 18.00 25.19 5.16 9 Leaflet length (cm) Mean 10.70 14.57 10.79 12.50 8.81 13.58 10.64 16.17 SD 1.44 1.15 1.02 0.00 1.13 1.56 1.32 0.23 COV 13.46 7.89 9.45 0.00 12.83 11.49 12.41 1.42 10 Leaflet width (cm) Mean 4.95 6.93 5.21 5.46 4.55 7.19 5.58 7.00 SD 0.98 0.68 0.82 0.00 0.65 0.55 1.05 0.00 COV 19.79 9.81 15.74 0.00 14.28 7.65 18.82 0.00 11 Number of leaflets Mean 9.29 9.73 8.80 9.00 7.65 8.17 8.00 8.00 SD 1.32 2.25 0.45 0.00 0.82 0.87 1.15 1.41 COV 14.21 23.12 5.11 0.00 10.72 10.65 14.37 17.62 Table 3 - Average inter- and intra-cluster distance among various clusters in walnut germplasm Clusters I II III IV V VI VII VIII I 1.658 II 3.535 1.951 III 2.295 3.975 1.497 IV 10.411 10.528 8.795 0.000 V 1.821 4.953 2.505 10.048 1.396 VI 3.159 2.115 2.689 9.396 3.875 1.657 VII 3.870 4.898 2.984 9.037 3.536 3.388 2.171 VIII 7.505 5.835 6.356 8.920 7.931 5.081 5.220 1.373 Bold figures represent intra-cluster distances. 127 tion came from plant height, while the minimum eigen roots value 0.045 and percent contribution towards diver- sity (0.41%) was observed for the eleventh component, with a maximum contribution from TCSA. The percent variation explained by the first seven components was 95.95 (Table 5). The genetic diversity among genotypes could be due to various factors, like heterogeneity, genetic architecture of populations and developmental traits, as described by (Murty and Arunachalam, 1966). Rao et al. (2003) re- ported that geographical distribution and genetic diversity are correlated and concluded that eco-geographically dif- ferent cultivars also differ from each other genetically. A clustering pattern similar to our pattern was reported in walnut (Pandey and Tripathi, 2007) and hazelnut (Sriv- astava et al., 2010). Sardana et al. (1997) observed that cluster means reveal the inner diversity in the material un- der study. De et al. (1988) proposed that traits contributing the most towards the D2 values should be given priority in choosing a cluster for further selection and choice of par- ents for hybridization. Based on the present findings of genetic divergence and its component analysis it can be concluded that inter- crossing between genotypes of genetically diverse clusters that show a superior mean outcome may be helpful for ob- taining desirable segregates. In the case of walnut germ- plasm the highest genetic diversity was registered between clusters II and IV. Comparison of the cluster means for 11 characters indicated that the studied traits considerably differed between the clusters. Therefore, it is suggested to cross ‘Howard’ with ‘Chamba Selection 123’ and with ‘Chandler’ as these accessions are among the most distant clusters, with a high mean outcome, in order to get desir- able transgressive segregates. References BARUA U., SHARMA R.K., 2003 - Estimation of genetic diver- gence in apple (Malus x domestica Borkh.). - Indian Journal of Plant Genetic Research, 16: 4-6. DE R.M., SEETHARAMAN R., SINHA M.T., BANARJEE S.P., 1988 - Genetic divergence in rice. - Indian Journal of Genetics, 48: 189-194. GUPTA S.K., 1999 - Characterization and evaluation of wal- nut (J. regia L.) germplasm accessions. - M.Sc. Thesis, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni- Solan, India. KAUSHAL R.K., SHARMA S.D., 2005 - Non-hierarchical Eu- clidean cluster analysis studies in seedling trees of pecan [Carya illinoensis (Wang) K. Koch.] growing in Himachal Pradesh. - Acta Horticulturae, 696: 97-101. MURTY B.R., ARUNACHALAM V., 1966 - The nature of di- vergence in relation to breeding system in crop plants. - In- dian Journal of Genetics, 26: 188-198. PANDEY G., TRIPATHI A.N., 2007 - Estimation of genetic di- vergence in walnut. - Indian Journal of Horticulture, 64(4): 399-401. PANSE V.G., SUKHATME P.V., 1995 - Statistical methods for agricultural workers. - ICAR Pub., New Delhi, pp. 296. RAO C.R., 1952 - Advanced statistical method in biometrical research. - John Wiley & Sons, New York, pp. 390. RAO E.S., VERMA V.K., MUNSHI A.D., 2003 - Breeding po- tential of cucumber (Cucumis sativus L.) genotypes using D2 analysis. - Indian Journal of Horticulture, 60(1): 53-58. SARDANA S., BORTHAKUR D.N., LAAKANPAL T.N., 1997 - Genetic divergence in rice germplasm of Tripura. - Oryza, 34: 201-208. SHARMA O.C., 1999 - Studies on variability and selection of superior Persian walnut (Juglans regia L.) seedling trees in Himachal Pradesh. - Ph.D. Thesis, Dr. Y.S. Parmar Univer- Table 5 - Eigen vectors, eigen roots and percent variation explained by growth and foliage characters of walnut germplasm S. No. Characters Eigen roots Per cent variation Eigen vectors 1 2 3 4 5 6 7 8 9 10 11 1 Plant height (m) 4.332 39.38 0.131 0.180 0.239 0.147 0.192 0.415 0.419 0.404 0.432 0.369 0.035 2 Trunk girth (cm) 3.008 27.34 0.458 0.490 0.293 0.456 0.295 -0.145 -0.126 -0.195 -0.169 -0.196 -0.163 3 Plant spread (cm) 1.155 10.50 -0.202 -0.228 0.424 -0.383 0.496 -0.128 0.018 -0.037 -0.042 0.068 -0.557 4 TCSA (cm2) 1.047 9.52 -0.059 -0.132 0.371 -0.170 0.326 0.211 0.099 -0.153 -0.129 -0.354 0.697 5 Plant volume (cm3) 0.424 3.85 -0.006 -0.018 -0.379 0.051 0.579 -0.023 -0.483 0.308 -0.160 0.337 0.225 6 Leaf length (cm) 0.347 3.15 0.818 -0.293 -0.075 -0.391 -0.025 -0.076 0.057 -0.158 0.037 0.213 0.066 7 Leaf width(cm) 0.243 2.20 -0.054 0.125 0.512 -0.070 -0.326 -0.468 -0.111 0.276 -0.231 0.418 0.270 8 Leaf area (cm2) 0.191 1.74 -0.199 0.026 -0.206 0.179 0.242 -0.426 0.469 -0.506 0.101 0.357 0.180 9 Leaflet length (cm) 0.118 1.07 0.110 -0.047 -0.209 -0.024 0.141 -0.452 0.446 0.565 -0.110 -0.432 -0.002 10 Leaflet width (cm) 0.091 0.83 -0.013 0.060 0.073 -0.090 0.049 -0.362 -0.354 0.001 0.813 -0.217 0.126 11 Number of leaflets 0.045 0.41 -0.060 0.742 -0.192 -0.627 0.024 0.058 0.074 -0.056 -0.056 -0.003 0.021 Percent variation explained by first 7 components = 95.9517 128 sity of Horticulture and Forestry, Nauni-Solan, India. SHARMA O.C., 2002 - Selecting walnuts from seedling tress populations in some villages of district Sirmour (H.P.). - M.Sc. Thesis, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni-Solan, India. SHARMA O.C., MURKUTE A.A., KANWAR M.S., 2010 - As- sessing genetic divergence in seedling trees of Persian wal- nut (Juglans regia L.). - Indian Journal of Agricultural Sci- ences, 80(5): 360-363. SHARMA O.C., SHARMA S.D., 2005 - Intra specific divergence in seedling trees of Persian walnut (Juglans regia) for metric tree, foliage and floral characters in Himachal Pradesh, India. - Plant Genetic Resources Newsletter, 144: 45-53. SRIVASTAVA K.K., ZARGAR K.A., SINGH S.R., 2010 - Ge- netic divergence among Corylus colurna genotypes based on morphological characters of hazelnut. - Biodiversity Re- search and Conservation, 17: 13-17. THAKUR B.S., SHARMA O.C., MEHTA K., SHARMA D.P., REHALIA A.S., 2005 - Genetic divergence in almond (Prunus amygdalus Batsch) growing in Kinnaur, Himachal Pradesh - Acta Horticulturae, 696: 69-74. THAKUR D., 1993 - Genetic variability in bearing seedling walnuts (J. regia L.) in Kullu valley. - M.Sc. Thesis, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni-So- lan, India.