Atlas Journal of Biology 2018, pp. 575–582 doi: 10.5147/ajb.v0i0.179 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) Morphological and Pomological Variability Analysis of Wal- nut (Juglans regia L.) Genetic Resources from the Middle and High Atlas of Morocco Ghizlane Kabiri, Said Bouda*, Mohamed Elhansali, and Abdelmajid Haddioui Laboratory of Biotechnologies and Valorization of Plant Gnetic Resources, University of Sultan Moulay Slimane, Faculty of Sciences and Techniques, P.B. 523, Beni Mellal, Morocco Received: October 12, 2018 / Accepted: December 1, 2018 __________________________________________________ * Corresponding author: saidbouda@yahoo.fr 575 Abstract The walnut (Juglans regia L.) is conducted yet as a tradi- tional fruit crop in different environments of Moroccan mountains. Besides, little data is available on its genetic diversity. Thus, twenty-one morphological and pomologi- cal characters of 11 Moroccan accessions were used to as- sess phenotypic variation of this species. Significant differ- ences were found between accessions for the most examined traits, indicating a high phenotypic diversity. Multivariate analyses lead to identify three groups of accessions. The first group composed by the accessions of both Middle and High Atlas Mountain with high weight and percentage of kernel and low thickness of shell. The second group is made of ac- cessions from the High Atlas Mountain characterised by large leaf, high nut dimension and large nut size. The third group is composed of two accessions; one belongs to the Middle Atlas Mountain and the other originating from High Atlas Mountain and characterized by small leaf and nut. Then, the structuration of accessions in three groups was not correlated to the mountain range type. So, this study showed a significant phenotypic heterogeneity between the eleven studied accessions which opens the way for the strat- egies for their conservation and the selection of efficient genotypes with the desired traits. Keywords: Walnut; Juglans regia L; Morocco’s accessions; Leaf variability; Fruit variability. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creative- commons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction The walnut species (Juglans regia L.) is one of the most widely cultivated and economically important species of edible nuts in the temperate regions of the world (McGranahan and Leslie, 1990). This species is originated from areas of Central Asia (Amiri et al., 2010) and its cultivation and production were centered in USA and China (Angmo et al., 2013). Throughout history, the nuts have been a staple food providing energy, pro- tein, essential fatty acids, vitamins and minerals. They are also being investigated for their potential health benefits (Dreher et al., 1996). The walnut tree has a great nutritional value and the noble wood. Moreover, walnuts have significant economic value and medicinal importance for human health, because of their biochemical composition of polyunsaturated fatty acids, especially linoleic acid and alpha-linolenic and protein value (Savage et al., 2001). The first introduction of this species, as a traditional fruit crop, to the great Maghreb was attributed to the Romans (Ger- main, 1992). In Morocco, walnut trees cover an area of 7600 Ha and are cultivated in different environments (Lansari et al., 2001). The trees can be found in humid and warm conditions, in the Rif and Atlas Mountains (High and Middle Atlas) and in arid regions in Southeastern Morocco. More than half of the plantings resulted from the prevailing way of seed propaga- tion practiced by farmers, since grafting is less adopted (Lan- sari et al., 2001). Genetic research on Moroccan Juglans regia has received little attention and few studies have concerned its genetic diversity. Only one study of phenotypic variability of few South-Eastern populations was carried out by Kodad et al. (2014). Consequently, it would be very interesting to find more A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) discriminating criteria that could provide additional information on genetic diversity of Moroccan walnut populations. Thus, the present study was conducted to characterize and evaluate the genetic variability of walnut accessions using some morpholog- ical and pomological traits and to look for any space structuring of such accessions according to their distribution on the Atlas Mountains in Morocco. Materials and Methods Plant Material and Phenotyping Traits During September 2014, fresh leaves and mature nuts were collected from eleven Moroccan accessions representing the main cropping area of walnut. Table 1 and Figure 1 present the accessions investigated as well the characteristics of their geo- graphical and ecological origins. For each accession, 10 trees were randomly chosen and sampled. From each tree, ten devel- oped leaves and 20 nuts were collected from different sites and elevation of the tree to be subject of observations and measure- ments considered in the present study. 576 According to the IPGRI and UPOV descriptors (IPGRI, 1994; UPOV, 1999), 31 parameters related to leaves and fruits (Table 2) were considered, following instructions given by the IPGRI and UPOV descriptors. All traits on the kernel have been measured one month after harvest when the water content was below 8% (UPOV, 1999). Statistical Analyses The obtained data was submitted to Analysis of variance (ANOVA) to search for any significant differences among ac- cessions for the parameters analyzed. When significant dif- ferences occurred among accessions, a LSD test was used to determine if there were significant differences between indi- vidual accessions. Association between traits was established using Pearson correlation coefficient. All these analyses were performed by using Statistical Analysis System program (SAS, 1999). Accessions ordination and classification were performed using the principal component analysis (PCA)( XLSTAT, 2015) and the hierarchical cluster analysis (Statistica StatSoft, 1997). The PCA and the cluster analyses were carried out on the matrix of mean values of measured characters. A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) Figure 1. Morocco map showing sampling sites of the analyzed walnut accessions. Table 1. Geographic origin and ecological characteristics of the 11 walnut (Juglan rejia L.) accessions. Accessions Code Geographic Origin Altitude (m) Latitude N Longitude W Zone Rainfall Average (mm) Aghbala AGH 32 Km North East of Aghbala 1673 32°32’ 5°39’ Middle Atlas 450 Naour NAO Central Naour 1300 32°29’ 5°58’ Middle Atlas 600 Taghzirte TAG 12 Km East of Tagzirte 650 32 26 6° 12’ Middle Atlas 700 Ait Bougamez ABZ Ait Bougamez Centre 1996 31°38’ 6° 28’ High Atlas 580 Ait M’hamed AMD 20 Km South East of Azilal 1728 31° 25’ 2° 28’ High Atlas 450 Demnate DEM 3 km South East of Demnate 932 31° 43’ 6° 58’ High Atlas 350 Imlil IML 17 km South of Asni 1763 31° 8’ 7° 55’ High Atlas 459 Anougal ANG 40 km South of Amzmiz 1569 31° 9’ 8° 15’ High Atlas 681 Beram BER 5 km South of Midelt 1521 32° 40’ 4° 44’ High Atlas 210 Amouguer AMG 40 km West of Rich 1569 32° 12’ 5° 8’ High Atlas 250 Tabrijjate TBR 70 km East of Imilchil 1831 32° 16’ 4° 56’ High Atlas 319 577 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) -Leaf traits Width of Pad on Suture WPS Leaf Length LL Prominence of Pad on Suture PPS Leaf Width LW Shape of Base Perpendicular to Suture SBPS Number of Leaflet Nlet Shape of Apex Perpendicular to Suture SAPS Leaflet Shape LetS Prominence of Apical Tip PAT Leaflet Width LetW Structure of Surface of Shell SSSh Leaflet Length LetL Shell Colour ShC Leaflet Margin LetM Shell Strength ShS Leaf Colour LC Adherence of Two Halves of Shell ATHSh Rachis Colour RC Thickness of Shell TSh -Nut traits -Kernel traits Nut Shape in Longitudinal section through Suture NSLS Ease of Removal of Kernel ERK Nut Shape in Longitudinal section Perpendicular to Suture NSLPS Kernel Weight KW Nut Width NWth Kernel Percentage KP* Nut Length NL Kernel Fill KF Nut Weight NW Kernel Colour KC Nut: Position of Pad on Suture NPPS Kernel Flavor KFV Kernel percentage = Kernel weigh/nut weight*100 Table 2. Morphological and pomological traits analyzed in the 11 walnut accessions. Results Mean values of morphological and pomological characters measured and their standard deviations are presented in Table 3. Results of analysis of variance indicated significant differences between accessions for all parameters except number of leaflets and leaflet margin. Leaf Morphological Traits Morphological traits related to leaf show large variability between accessions. In fact, leaf length varied among acces- sions from 37.98 cm for Aghbala to 44.25 cm for Anougal with an average of 41.52cm, the leaf width, with a general mean of 25.85cm, ranged from 24.46 cm for Taghzirte to 27.17 cm for Beram and the number of leaflets scaled from 7.38 for Naour to 8.75 for Taghzirte with an average of 8.24. In addition, leaflet length, having an average of 10.75 cm, varied from 9.37 cm for Taghzirte to 11.16 cm for Imlil. With a general mean of 5.48 cm, the highest value of leaflet width was recorded in Tabrijjate (6.22 cm) and the lowest was observed in Taghzirte (4.98 cm). However, the number of leaflets per leaf has varied narrowly among accessions, within a range of 7.3 for Naour to 8.75 for Taghzirte, and averaged 8.24. Furthermore, with an entire leaf- let margin for almost all, the accessions did not show any differ- ence for this trait. In general, the color of leaf was green with a rachis color varied between green, yellow to red. Fruit Pomological Traits The nut weight for the accessions tested varies within the range of 8.26 g for Aghbala to 10.20 g for Tabrijjate, with an average of 9.48 g. The average nut length was 35.46 mm and ranged from 32.08 mm for Demnate to 38.11 mm for Imlil. For nut width, the lowest value was obtained for Demnate (27.39 mm) and the highest for Tabrijjate (31.50 mm) with a mean of 30.10 mm. The shell thickness of the studied accessions in this investigation varied between 1.33 mm for Amouguer and 1.83 mm for Ait M’hamed with a value of 1.60 mm as average. Regarding the kernel weight, the highest value was observed in Amouguer (4.13 g), while the least was obtained for Aghbala (2.86 g), with a mean of 3.71g. The kernel percentage ranged from 33.65% in Demnate to 43.76% in Amouguer and averaged 38.28%. For nut shape, in general, it was elliptic board with a medium shell color and intermediate shell strength. Concerning kernel color, 9% of the walnut accessions were revealed with light kernel, 36% with amber kernel and 55% with light am- ber kernel. For kernel flavor, it was revealed satisfactory for all walnut accessions. For the remaining analyzed traits, they have varied widely among accessions indicating the existence of high pomological variability in Moroccan walnut germplasm. Association Between Parameters Studied Correlation among all morphological and pomological traits is summarized in table 4. The strongest negative correlations were observed between, on one hand, kernel weight (KW) and, on the other hand, leaf length, (LL), leaf width (LW), leaflet length (LetL), nut length (NL), nut weight (NW), shape of base perpendicular to suture (SBPS) and kernel colour (KC), with respective coefficients of -0.95, -0.93, -0.93, -0.92, -0.91, -0.95 and -0.96. However, strong positive correlations were found between kernel weight (KW) and number of leaflet (Nlet) (r=0.97), nut shape (NSLPS) (r=0.97), prominence of apical A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) 578 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) Ta bl e 3. M ea n va lu es a nd st an da rd d ev ia tio n fo r l ea f, nu t a nd k er ne l t ra its m ea su re d in 1 1 M or oc ca n w al nu t a cc es si on s. Tr ai ts Ta gh zi rt e Ta br ijj at e Am ou gu er An ou ga l Ai t M ha m ed Im lil Be ra m Ag hb al a N ao ur Ai t b ou ga m ez D em na te M ea n F LL ( cm ) 39 .1 4± 3. 19 a 42 .1 4± 3. 76 b 39 .3 9± 4. 14 a 44 .2 5± 6. 51 b 43 .3 5± 5. 54 b 44 .1 4± 5. 88 b 42 .8 0± 4. 19 b 37 .9 8± 3. 85 a 39 .2 8± 4. 06 a 40 .1 7± 4. 75 a 44 .0 8± 3. 12 a 41 .5 2 4. 68 ** * LW ( cm ) 24 .4 6± 2. 20 ab 25 .9 1± 4. 29 a 27 .1 1± 3. 06 c 25 .4 7± 3. 95 a 24 .9 0± 2. 99 a 27 .0 3± 3. 67 c 27 .1 7± 3. 09 c 24 .7 ±4 .1 1a 25 .5 0± 6. 42 b 25 .4 6± 3. 20 a 26 .6 5± 4. 09 c 25 .8 5 6. 23 ** * N le t 8. 75 ± 0. 62 b 7. 5 ± 1. 06 a 8. 05 ±1 .0 3a c 8. 34 ±0 .9 0b c 8. 62 ±0 .6 8b 8. 56 ±0 .9 8b 8. 67 ±0 .9 4b 7. 6± 1. 03 a 7. 38 ±1 .6 6a 8. 37 ±0 .8 3b c 8. 60 ± 0 .7 7b 8. 24 1. 77 Le tS 2. 46 ±0 .7 6a b 2. 95 ±0 .2 0c 2. 52 ±0 .6 4b 2. 42 ±0 .6 5a b 2. 77 ±0 .4 2b c 2. 93 ±0 .2 4c 2. 52 ± 0 .6 6b 2. 20 ±0 .8 4a 2. 3± 0. 59 ab 2. 67 ±0 .7 0b c 2. 93 ±0 .3 6c 2. 61 3. 33 ** * Le tW (c m ) 4. 98 ±0 .7 7a 6. 22 ±0 .9 6c 5. 31 ±0 .6 7a b 5. 75 ±1 .5 1b c 5. 59 ±0 .7 8b 5. 49 ±0 .8 6b 5. 34 ±0 .8 0a b 5. 35 ±0 .9 5a b 5. 56 ±1 .0 2b 5. 27 ±0 .8 1a b 5. 40 ±1 .2 5a b 5. 48 4. 33 ** * Le tL (c m ) 9. 37 ±1 .4 2a 11 .0 2± 1. 68 b 10 .7 8± 1. 37 b 10 .7 2 ± 1. 84 b 10 .5 1± 1. 14 b 11 .1 6± 1. 57 b 10 .5 3± 1. 56 b 10 .4 8± 1. 74 b 10 .6 5± 2. 01 b 10 .5 9± 1. 40 b 10 .4 9± 1. 65 b 10 .5 7 3. 09 ** Le tM 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1. 00 1. 22 LC 5. 68 ±1 .0 9b 5. 66 ±0 .9 6b 4. 78 ±1 .0 1a 5. 45 ±1 .2 9b 5± 00 a 4. 81 ±1 .5 5a 5± 00 a 5. 33 ±1 .0 6b 5± 00 a 5. 12 ±0 .4 9a 5± 00 a 5. 17 3. 75 ** * RC 3. 68 ±1 .3 0a 3. 33 ±0 .9 6a 4. 63 ±0 .7 8c 3± 00 a 4. 18 ±1 .0 0b c 4. 43 ±1 .8 4b c 4. 11 ±1 .0 0b 4± 1. 36 b 6. 86 ±0 .5 0d 4. 06 ±1 .0 1b 3. 06 ±0 .3 6a 4. 12 4. 14 ** * N SL S 6. 32 ±1 .7 4c d 4. 97 ± 1. 97 a 6. 43 ±2 .0 2c 5. 27 ±1 .9 6a 5. 78 ±2 .1 3c 5. 34 ±2 .5 8b 5. 86 ±1 .9 2c 6. 57 ±1 .1 3c 5. 57 ±2 .0 5a 6. 23 ±1 .7 0c 6. 97 ±1 .6 5d 5. 95 10 .3 4* ** N SL PS 6. 65 ±0 .7 2d 4. 77 ± 2. 39 a 6. 70 ±1 .3 0c 5. 94 ±1 .5 6c 5. 54 ±2 .1 1c 5. 01 ±2 .4 4b 6. 09 ±1 .7 1c 6. 65 ±1 .1 0c 5. 54 ±2 .2 5a 6. 35 ±1 .8 7d c 6. 26 ±1 .8 1d 5. 97 6. 67 ** * N W th (m m ) 29 .6 7± 2. 47 b 31 .5 0± 3. 17 a 29 .1 8± 2. 69 b 31 .2 4± 2. 74 c 31 .3 6± 4. 60 a 31 .3 9± 2. 55 c 29 .7 7 ± 3. 29 ab 29 .5 0± 1. 95 b 30 .1 3± 2. 87 b 30 .3 0± 1. 94 a 27 .3 9± 8. 81 a 30 .1 0 19 .8 7* ** N L (m m ) 34 .1 3± 2. 81 b 35 .0 7± 3. 62 bc 35 .1 1± 3. 53 c 37 .0 2± 3. 36 d 36 .4 5± 4. 63 a 38 .1 1± 3. 27 e 34 .8 4± 4. 36 ac 35 .2 3± 2. 12 ac 36 .0 8± 3. 77 a 36 .7 4± 3. 13 d 32 .0 8± 10 .8 2a 35 .4 6 17 .2 7* ** N W (g ) 9. 24 ±1 .6 1c 10 .2 0± 2. 11 b 9. 42 ±2 .8 3c 9. 84 ±2 .1 4b 10 .1 4± 4. 00 b 9. 28 ±2 .1 2c 9. 71 ±2 .6 4b 8. 26 ±2 .0 5b 9. 79 ±2 .9 4b 9. 54 ±1 .5 5b 8. 91 ±3 .5 1b 9. 48 7. 61 ** * N PP S 2. 15 ±0 .7 5d 1. 58 ±0 .7 5b 1. 61 ±0 .9 1b 1. 22 ±0 .4 8a 1. 73 ±0 .6 8b 1. 74 ±0 .7 6b 1. 86 ±0 .7 7c 1. 96 ±0 .9 3c 2. 15 ±0 .6 5d 2. 44 ±0 .7 9e 2. 03 ±1 .0 4c 1. 86 13 .1 2* ** W PS 3. 94 ±0 .9 9b 4. 68 ±0 .8 0e f 4. 84 ±0 .7 3f 5. 24 ±0 .8 6g 4. 60 ±1 .5 3e 5. 04 ±0 .9 4g 4. 36 ±0 .9 7c d 4. 34 ±0 .9 4d 4. 12 ±1 .3 1c 3. 59 ±0 .9 3a 3. 70 ±0 .9 5a 4. 40 8. 95 ** * PP S 4. 32 ±1 .1 0a 4. 94 ±0 .5 8c 4. 60 ±1 .1 3b 4. 86 ±0 .8 6c 4. 64 ±0 .7 6b 5. 02 ±0 .9 2c d 5. 26 ±1 .1 2d 4. 98 ±0 .7 7c 4. 19 ±0 .9 9a 4. 48 ±0 .9 6b 4. 51 ±1 .3 7b 4. 71 6. 2* ** SB PS 1. 77 ±0 .5 0b 2. 15 ±0 .6 2d 1. 45 ±0 .5 5a 2. 19 ±0 .5 1d 2. 02 ±0 .4 7c 2. 20 ±0 .6 3d 1. 78 ±0 .7 1b 1. 54 ±0 .7 3a 2. 06 ±0 .9 3c 1. 72 ±0 .6 9b 1. 82 ±1 .2 0a 1. 88 9. 25 ** * SA PS 3. 06 ±0 .6 4d 2. 84 ±0 .5 8b c 2. 68 ±0 .4 6b 2. 69 ±0 .5 4b 2. 41 ± 0 .5 5a 2. 24 ±0 .8 0a 2. 85 ±0 .8 5c 2. 25 ±0 .5 3a 2. 17 ±0 .8 7a 2. 62 ±0 .7 3b 3. 03 ±0 .9 4c 2. 62 11 .3 ** * PA T 3. 05 ±0 .6 4a 3. 87 ±0 .9 9b 3. 54 ±0 .9 5a 3. 94 ±1 .0 9b 3. 72 ±1 .0 3a b 4. 9± 0. 77 d 3. 99 ±1 .2 0b 4. 4± 1. 16 c 3. 58 ±0 .9 2a 4. 02 ±1 .1 0b 3. 89 ±1 .0 9b 3. 90 3. 05 ** * SS Sh 2± 00 bc 2. 38 ±0 .4 9d 1. 76 ±0 .6 5a 2. 35 ±0 .6 4d 2. 62 ±0 .8 6e f 2. 51 ±0 .6 4e 2. 70 ±0 .8 1f 2. 65 ±0 .5 5e f 2. 16 ±0 .3 6c 2. 14 ±0 .3 7c 2. 10 ±0 .3 0b c 2. 31 18 .3 2* ** Sh C 6. 42 ±1 .4 8f 4. 40 ±2 .4 8c d 3. 96 ±1 .7 1b c 4. 15 ±1 .1 9c 5. 15 ±2 .2 0e 5. 25 ±1 .4 5e 3. 86 ±1 .4 5b 3. 88 ±1 .6 6b 4. 40 ±1 .9 9d 3. 37 ±1 .6 5a 4. 97 ±0 .6 1e 4. 53 40 .7 9* ** Sh S 4. 70 ±0 .7 7b 5. 48 ±1 .1 5d 4. 68 ±0 .9 3b 5. 53 ±1 .3 5d 5. 27 ±0 .9 1d 4. 34 ±1 .1 0a 5. 32 ±0 .8 8c d 5. 1± 0. 99 c 5. 18 ±0 .7 0c d 4. 33 ±1 .0 0a 5. 71 ±1 .8 3d 5. 06 7. 19 ** * AT H Sh 4. 65 ±0 .7 8b 5. 24 ±0 .7 3d 4. 47 ±0 .8 8b 4. 18 ±1 .3 0a 5. 21 ±1 .0 2d 4. 30 ±1 .2 5a b 5. 15 ±1 .1 5c d 5. 12 ±1 .0 1c d 5. 5± 0. 99 e 4. 35 ±1 .0 0a b 4. 96 ±0 .8 4c 4. 83 9. 84 ** * TS h (m m ) 1. 57 ±0 .3 2c 1. 71 ±0 .3 0d 1. 33 ±0 .4 0a 1. 69 ±0 .4 6d 1. 83 ±0 .5 1e 1. 55 ±0 .3 8c 1. 66 ±0 .4 1d 1. 50 ±2 .8 8b c 1. 61 ±0 .3 6c d 1. 44 ±0 .3 8b 1. 72 ±0 .3 4d 1. 60 4. 07 ** * D RK 4. 3± 1. 01 a 5. 7± 1. 22 a 4. 56 ±0 .9 8b 5. 28 ±1 .1 8d 4. 87 ±1 .3 6c 4. 7± 1. 34 bc 4. 66 ±1 .4 1b c 4. 3± 1. 16 a 4. 79 ±1 .1 4c 4. 25 ±0 .9 9a 5. 24 ±1 .3 2d 4. 79 8. 58 ** * K W (g ) 3. 71 ±1 .1 2b c 4. 02 ±1 .1 5c e 4. 13 ±1 .4 8e 3. 62 ±1 .3 8b c 3. 61 ±1 .3 6b c 3. 87 ±1 .3 6c 3. 78 ±1 .2 7b c 2. 86 ±1 .1 1a 3. 77 ±1 .6 9b c 4. 01 ±1 .1 2c e 3. 45 ±1 .2 5b 3. 71 5. 56 ** * K P (% ) 39 .4 5± 8. 94 cd 38 .7 2± 7. 23 c 43 .7 6± 9. 74 e 35 .4 9± 9. 40 ab 36 .7 1± 11 .1 7b 41 .1 6± 10 .2 3d 38 .8 ±8 .1 4c 33 .6 1± 10 .0 3a 37 .7 3± 9. 67 c 42 .0 1± 8. 42 de 33 .6 5± 9. 90 ab 38 .2 8 5. 3* ** K F 6. 39 ±1 .1 7d 6. 45 ±1 .2 4d 5. 82 ±1 .5 0b c 5. 26 ±1 .5 8a 6. 31 ±1 .3 3c d 5. 60 ±1 .6 2b 6± 1. 37 c 5. 56 ± 1 .6 8b 6. 27 ±1 .1 2d 6. 45 ±1 .1 5d 6. 24 ±1 .3 4c d 6. 03 9. 55 ** * K C 3. 6± 0. 58 2. 72 ±0 .9 6 3. 32 ±0 .6 6 3. 21 ±0 .7 6 2. 94 ±0 .7 8 3. 36 ±0 .8 9 3. 22 ±0 .6 9 2. 57 ±0 .9 9 3. 25 ±0 .8 2 2. 73 ±0 .7 8 3. 25 ±0 .5 1 3. 11 26 .0 7* ** K FV 1. 06 ±0 .2 5 1. 04 ±0 .2 0 1. 05 ±0 .2 2 1. 30 ±0 .4 6 1. 06 ±0 .2 5 1. 15 ±0 .3 6 1. 07 ±0 .2 6 1. 31 ±0 .4 6 1. 02 ±0 .1 4 1. 02 ±0 .1 4 1. 08 ±0 .2 8 1. 11 7. 83 ** * Si gn ifi ca nc e le ve l: ** * :P < 0. 00 1; * *: P < 0. 01 ; * : P< 0. 05 . D iff er en t l et te rs n ot e si gn ifi ca nt d iff er en ce s ( LS D a t 0 .0 5 le ve l). In b ol d ar e m in im um a nd m ax im um v al ue s A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) 579 LL LW N let Le tS Le tW Le tL Le tM LC R C N SL S N SL PS N W th N L N W N PP S W PS PP S SB PS SA PS PA T SS Sh Sh C Sh S A TH Sh TS h D R K K W K P K F K C LW 0, 97 ** * N let -0 ,9 5* ** - 0, 93 ** * Le tS -0 ,9 5* ** - 0, 93 ** * 0, 98 ** * Le tW -0 ,5 9* ** - 0, 58 ** * 0, 73 2* ** 0 ,7 1* ** Le tL 0, 96 ** * 0, 95 ** * -0 ,9 3* ** - 0, 93 ** * -0 ,5 6* ** Le tM -0 ,8 3* ** - 0, 82 ** * 0, 85 ** * 0, 84 ** * 0, 62 ** * -0 ,8 2* ** LC 0, 15 ** * 0, 14 ** * -0 ,1 0* ** - 0, 13 ** * -0 ,0 2 0, 17 ** * -0 ,0 7 R C -0 ,3 6* ** - 0, 32 ** * 0, 38 ** * 0, 42 ** * 0, 29 ** * -0 ,3 7* ** 0, 24 ** * -0 ,2 4* ** N SL S -0 , 0 4 0, 01 0, 03 0, 05 0, 05 -0 ,0 3 -0 ,0 6 -0 ,0 7 0, 48 ** * N SL PS -0 ,9 5** * -0 ,9 3* ** 0, 97 ** * 0, 97 ** * 0, 69 ** * -0 ,9 3* ** 0, 84 ** * -0 ,1 3* ** 0, 38 ** * 0, 05 N W th -0 ,5 9* ** - 0, 57 ** * 0, 68 ** * 0, 7* ** 0, 64 ** * -0 ,5 6* ** 0, 56 ** * 0, 00 1 0, 36 ** * 0, 06 0, 68 ** * N L 0, 95 ** * 0, 93 ** * -0 ,9 1* ** - 0, 93 ** * -0 ,5 1* ** 0, 93 ** * -0 ,8 0* ** 0, 19 ** * -0 ,3 8* ** -0 ,0 1 -0 ,9 1* ** - 0, 47 ** * N W 0, 92 ** * 0, 90 ** * -0 ,9 0* ** - 0, 91 ** * -0 ,5 8* ** 0, 92 ** * - 0 ,8 1* ** 0, 17 ** * -0 ,3 7* ** -0 ,0 2 -0 ,9 1* ** - 0, 49 ** * 0, 94 ** * N PP S -0 ,7 2* ** - 0, 72 ** * 0, 73 ** * 0, 73 ** * 0, 52 ** * -0 ,7 1* ** 0, 83 ** * -0 ,0 6 0, 15 ** * -0 ,0 8* 0, 74 ** * 0, 52 ** * -0 ,6 9* ** - 0, 71 ** * W PS -0 ,2 3* ** - 0, 21 ** * 0, 25 ** * 0, 24 ** * 0, 20 ** * -0 ,1 7* ** 0, 21 ** * 0, 41 ** * -0 ,0 5 -0 ,1 3* 0, 25 ** * 0, 29 ** * -0 ,1 7* ** - 0, 13 ** * 0, 22 ** * PP S -0 ,2 7* ** - 0, 23 ** * 0, 30 ** * 0, 34 ** * 0, 28 ** * -0 ,2 9* ** 0, 17 ** * -0 ,2 1* ** 0, 62 ** * 0, 33 ** * 0, 30 ** * 0, 30 ** * -0 ,2 9* ** - 0, 28 ** * 0, 09 * -0 ,0 9* SB PS 0, 98 ** * 0, 95 ** * -0 ,9 5* ** - 0, 95 ** * -0 ,6 0* ** 0, 95 ** * -0 ,8 3* ** 0, 16 ** * -0 ,3 8* ** -0 ,0 05 -0 ,9 5* ** - 0, 59 ** * 0, 95 ** * 0, 92 ** * -0 ,7 2* ** - 0, 21 ** * -0 ,2 6* ** SA PS -0 ,4 7* ** - 0, 45 ** * 0, 59 ** * 0, 56 ** * 0, 57 ** * -0 ,4 5* ** 0, 50 ** * 0, 01 0, 17 ** * -0 ,0 5 0, 57 ** * 0, 56 ** * -0 ,4 2* ** - 0, 45 ** * 0, 43 ** * 0, 25 ** * 0, 11 ** -0 ,4 4* ** PA T -0 ,9 4* ** - 0, 93 ** * 0, 96 ** * 0, 98 ** * 0, 67 ** * -0 ,9 2* ** 0, 83 ** * -0 ,1 5* ** 0, 43 0, 06 0, 96 ** * 0, 70 ** * -0 ,9 2* ** - 0, 90 ** * 0, 72 ** * 0, 23 ** * 0, 36 ** * -0 ,9 4* ** 0, 59 ** * SS Sh -0 ,7 9* ** - 0, 77 ** * 0, 84 ** * 0, 84 ** * 0, 74 ** * -0 ,7 8* ** 0, 70 ** * -0 ,1 5* ** 0, 38 ** * 0, 07 0, 84 ** * 0, 67 ** * -0 ,7 2* ** - 0, 76 ** * 0, 61 ** * 0, 17 ** * 0, 31 ** * -0 ,7 9* ** 0, 66 ** * 0, 87 ** * Sh C 0, 87 ** * 0, 85 ** * -0 ,8 4* ** - 0, 84 ** * -0 ,5 3* ** 0, 89 ** * -0 ,7 6 * ** 0, 17 ** * -0 ,3 3* ** -0 ,0 02 -0 ,8 4* ** - 0, 47 ** * 0, 85 ** * 0, 86 ** * -0 ,6 8* ** -0 ,1 4* * -0 ,2 4* ** 0, 88 ** * -0 ,3 4* ** - 0, 84 ** * -0 ,6 9* ** Sh S 0, 89 ** * 0, 87 ** * -0 ,8 5* ** - 0, 87 ** * -0 ,5 1* ** 0, 87 ** * -0 ,6 5* ** 0, 19 ** * -0 ,4 2* ** -0 ,0 9 * -0 ,8 6* ** - 0, 50 ** * 0, 88 ** * 0, 85 ** * -0 ,5 2* ** -0 ,1 4* * -0 ,3 4 * ** 0, 90 ** * -0 ,2 8* ** - 0, 86 ** * -0 ,7 0* ** 0 ,8 2* ** A TH Sh -0 ,8 7* ** - 0, 85 ** * 0, 87 ** * 0, 87 ** * 0, 54 ** * -0 ,8 4* ** 0, 76 ** * 0, 1* 0, 30 ** * -0 ,0 07 0, 87 ** * 0, 59 ** * -0 ,8 5* ** - 0, 81 ** * 0, 67 ** * 0, 43 ** * 0, 21 ** * -0 ,8 6* ** 0 ,5 1* ** 0, 88 ** * 0, 72 ** * -0 ,7 4* ** - 0, 76 ** * TS h -0 ,1 1* * -0 ,0 7 0, 15 ** * 0, 19 ** * 0, 15 ** * -0 ,1 6* ** 0, 03 -0 ,0 8* 0, 54 ** * 0, 37 ** * 0, 15 ** * 0, 2* ** -0 ,1 5* ** - 0, 15 ** * -0 ,0 1 -0 ,1 6* ** 0, 60 ** * -0 ,1 3* * 0, 07 0, 20 ** * 0, 19 ** * -0 ,1 3* * -0 ,2 1* ** 0, 07 5 D R K -0 ,3 8* ** - 0, 31 ** * 0, 37 ** * 0, 40 ** * 0, 23 ** * -0 ,3 8* ** 0, 23 ** * -0 ,1 5* ** 0, 52 ** * 0, 31 ** * 0, 37 ** * 0, 27 ** * -0 ,3 9* ** - 0, 35 ** * 0, 14 ** * 0, 05 0, 43 ** * -0 ,3 8* ** 0, 13 ** 0, 40 ** * 0, 33 ** * -0 ,3 4* ** - 0, 43 ** * 0, 33 ** * 0, 55 ** * K W -0 ,9 5* ** - 0, 93 ** * 0, 97 ** * 0, 97 ** * 0, 69 ** * -0 ,9 3* ** 0, 84 ** * -0 ,1 3 * * 0, 38 ** * 0, 05 0, 97 ** * 0, 67 ** * -0 ,9 2* ** - 0, 91 ** * 0, 74 ** * 0, 24 ** * 0, 29 ** * -0 ,9 5* ** 0 ,5 7* ** 0, 97 ** * 0, 84 ** * -0 ,8 4* ** - 0, 86 ** * 0, 88 ** * 0, 15 ** * 0, 37 ** * K P -0 ,9 5 * ** - 0, 93 ** * 0, 96 ** * 0, 98 ** * 0, 68 ** * -0 ,9 3* ** 0, 83 ** * -0 ,1 4* ** 0, 41 ** * 0, 06 0, 97 ** * 0, 70 ** * -0 ,9 2* ** - 0, 91 ** * 0, 73 ** * 0, 23 ** * 0, 34 ** * -0 ,9 5* ** 0 ,5 5* ** 0, 98 ** * 0, 84 ** * -0 ,8 4* ** - 0, 86 ** * 0, 87 ** * 0, 20 ** * 0, 41 ** * 0, 98 ** * K F 0, 94 ** * 0, 92 ** * -0 ,9 1* ** - 0, 92 ** * -0 ,5 0* ** 0, 92 ** * -0 ,7 9* ** 0, 19 ** * -0 ,3 7* ** -0 ,0 3 -0 ,9 1* ** - 0, 48 ** * 0, 97 ** * 0, 93 ** * -0 ,6 8* ** - 0, 17 ** * -0 ,3 0* ** 0, 94 ** * -0 ,4 2* ** - 0, 91 ** * -0 ,7 1* ** 0 ,8 4* ** 0, 88 ** * -0 ,8 4* ** - 0, 16 ** * -0 ,3 8* ** - 0, 90 ** * -0 .9 1* ** K C 0, 97 ** * 0, 95 ** * -0 ,9 6* ** - 0, 97 ** * -0 ,6 2* ** 0, 96 ** * -0 ,8 3* ** 0, 17 ** * -0 ,4 0* ** -0 ,0 3 -0 ,9 6* ** - 0, 58 ** * 0, 97 ** * 0, 95 ** * -0 ,7 2* ** - 0, 21 ** * -0 ,3 2* ** 0, 97 ** * - 0 ,4 9* ** - 0, 96 ** * -0 ,8 0* ** 0 ,8 8* ** 0, 89 ** * -0 ,8 7* ** - 0, 17 ** * -0 ,4 0* ** - 0, 96 ** * -0 ,9 6* ** 0, 97 ** * K FV 0, 81 ** * 0, 79 ** * -0 ,7 9* ** - 0, 81 ** * -0 ,4 8* ** 0, 79 ** * -0 ,5 8* ** 0, 15 ** * -0 ,3 7* ** -0 ,0 9* -0 ,8 0 * ** - 0, 41 ** * 0, 84 ** * 0, 84 ** * -0 ,4 7* ** -0 ,1 1* -0 ,3 5* ** 0, 81 ** * -0 ,3 5* ** - 0, 81 ** * -0 ,6 7 * ** 0 ,7 1* ** 0, 85 ** * -0 ,7 2* ** - 0, 22 ** * -0 ,4 0* ** - 0, 79 ** * -0 ,8 0* ** 0, 87 ** * 0, 86 ** * Ta bl e 4. C or re la tio n co effi ci en ts a m on g le af , n ut a nd k er ne l t ra its in th e st ud ie d ac ce ss io ns . Si gn ifi ca nc e le ve l: ** * :P < 0. 00 1; * *: P < 0. 01 ; * : P< 0. 05 . a bb re vi at io ns a s i n ta bl e 1. A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) 580 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) tip (PAT) (r=0.97) and kernel percentage (KP) (r=0.98). Fur- thermore, nut weight (NW) was revealed to be positively and strongly correlated with leaf length (LL) (r=0.92), leaf width (LW) (r=0.90), leaflet length (LetL) (r=0.92), nut length (NL) (r=0.94), shape of base perpendicular to suture (SBPS) (r=0.92), kernel fill (KF) (r=0.93) and kernel colour (KC) (r=0.95), while it has a negative correlation with number of leaflet (Nlet) (r=- 0.90), shape of nut (NSLPS) (r=-0.91), prominence of apical tip (PAT) (r=-0.90), kernel weight (KW) (r=-0.91) and kernel percentage (KP) (r=-0.91). Multivariate Analysis The eigenvalues obtained by principal component analysis (PCA) indicate that the first two components provide a good summary of the data. They explained 71.29% of the total varia- tion with each component explaining respectively 45.88% and 25.41%. The first component is defined negatively by weight and length of nut, color and fill of kernel and by length and width of leaf; and positively by weight and percentage of kernel and by shape and number of leaflet. The second component is positively correlated to thickness of shell, ease of removal the kernel and the color of rachis. Figure 2 illustrated accessions on the plot of the first two axis spaces, shows that accessions may constitute three groups. The first one is composed of two acces- sions of Middle Atlas Mountain: Naour (NAO) and Taghzirte (TAG) and two other accessions of High Atlas: Ait Bougamaz (ABZ) and Amouguer (AMG), characterized by high weight and percentage of kernel and low thickness of shell. The sec- ond group is made of five accessions of High Atlas Mountain namely Ait M’hamed (AMD), Imlil (IML), Beram (BER), Ta- brijjate (TAB) and Anougal (ANG), characterized with large leaf, high nut dimension and large nut size. The third group is composed of two accessions; one belongs to Middle Atlas (Aghbala, (AGH)) and the other arising from High Atlas (Dem- nate (DEM)) which are characterized by small leaf and nut. Hierarchical cluster analysis identified three distinct groups (Figure 3) confirming the PCA results. The first group is com- posed of Taghzirte (TAG) and Naour (NAO) accessions origi- nating from Middle Atlas Mountain and Amouguer (AMG) and Ait Bougamaz (ABZ) coming from High Atlas Mountain. The second group includes Ait M’hamed (AMD), Imlil (IML), Ba- rem (BAR), Tabrijjate (TBR) and Anougal (ANG) accessions, all belonging to High Atlas Mountain. The Demnate (DEM) and Aghbala (AGH) accessions represented the third group and seem to diverge significantly from the others. Figure 2. Plot on the two first principal components of 11 walnut accessions. Abbreviations as in table 1. 581 A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) Figure 3. Dendrogram of 11 Moroccan walnut accessions based on morphological and pomological traits. Abbreviations as in table 1. Discussion The results of this study show that the accessions studied could be a very interesting source of walnut genetic diversity. The majority of morphological traits of leaves showed a great difference among walnut accessions, especially for the length and width of leaf. Furthermore, the pomological traits exhib- ited the highest variability among accessions, which was estab- lished for most of nut and kernel traits. According to consulted literature, nut weight, kernel per- centage and color of kernel have been considered as the most important indices of quality and economic yield in walnut trees, which should be further investigated. In the Moroccan accessions studied, the nut weight varied from 8.26 to 10.20 g. Values that are within the consulted literature are found be- tween 6 and 16.89 g in Iranian genotypes (Arzani et al., 2008; Ghasemi et al., 2012; Ahandani et al., 2014), 7.46 and 15.21 g in Turkish genotypes (Karadag and Akça, 2011) and from 6.9 to 16.27 g in Romanian walnut (Cosmulescu, 2013). In addi- tion, Moroccan accessions have an average kernel percentage between 33.65% and 43.76%. These values are lower than those obtained for some walnut genotypes from Turkey (46.15 to 63.16%) (Karadag and Akça, 2011) and Iran (48 to 59%) (Ahandani et al., 2014). According to color of kernel, our result showed that 55% of the walnut accessions investigated were revealed with light amber kernel. This result is in accordance with that reported in Iranian genotypes by Arzani et al. (2008), finding 52% of genotypes with light amber kernel. In fact, the light kernel color is a primary breeding objective for walnut. On other hand, the shell thickness of the Moroccan accessions analysed in this work varied from 1.33 to 1.83 mm, which is in agreement with that obtained by Karadag and Akça (2011) (0.95 to 1.60 mm) and Akça et al. (2015) (1.11 to 2.33 mm) in Turkish walnut, but greater than that reported by Sharma et al. (2014) in Indian walnut (1.24 mm). These results showed a considerable phenotypic diversity in the Moroccan walnut germplasm in comparison to that of other countries. This variability may be due, first, to genotypic varia- tion or environmental conditions (Ghasemi et al., 2012). Sec- ond, walnuts species are monoecious and heterodichogamous, favoring outcrossing over selfing (Ebrahimi et al., 2016). Third, reproduce by seeds of this species causes a very important ge- netic variability that appears at the flowering period for pomo- logical characters, vigor of the tree and type of fructification, which allowed each geographic region to maintain a diverse population (Lansari et al., 2001). Besides, Diaz et al. (2005) stated that the geographic location and the growing conditions affect fruit and kernel traits of walnut populations from western Spain. Conclusion These results show that Moroccan walnut presents a high variability in comparison to that of others countries with simi- lar ecological conditions. This investigation showed significant differences among accessions, permitting discrimination be- tween them according to morphological traits related to leaf, nut and kernel. These findings should be exploited to select the best walnut accession with the wanted traits to be multiplied in order to encourage the agriculture and raise the production of walnut. Taking into account the relevance of high kernel weight, kernel percentage and low thickness of shell, the wal- A tla s J ou rn al o f B io lo gy - IS SN 2 15 8- 91 51 . P ub lis he d B y A tla s P ub lis hi ng , L P (w w w. at la s- pu bl is hi ng .o rg ) 582 nut accessions from Amouguer and Imlil should be considered as the first accessions to be useful as seed sources or multiplied vegetatively for walnut propagation in Morocco. It is obvious that survey of genetic diversity in Moroccan walnut using bio- chemical and molecular markers are projected and would be a complement for this study. References Ahandani EA, H Ramandi, J Sarmad, M samani, A Yavari and RA Ahandani (2014) Evaluation of Morphological Diversity among Some Persian Walnut Accessions (Juglans regia L.) In Guilan, Northern Iran. International Journal of Biotechnology 5(2): 21-30. Akca Y, Y Bilgen and S Ercisli (2015) Selection of Superior Persian Walnut (Juglans regia L.) from Seedling Origin in Turkey. 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