Genetic diversity assessment of ancient mulberry (Morus spp.) in Lebanon using morphological, chemical and molecular markers (SSR and ISSR)

Lebanon has ancient mulberry trees which are the remnants of the abundant orchards that dominated its lands during the nineteenth century. Lebanese mulberry germplasm has not been assessed yet. This study aims to collect local old rainfed mulberry accessions from different geographical regions and assess their diversity by using morphological and molecular mark­ ers (SSR and ISSR). Genetic diversity of 70 accessions of mulberry were evaluat­ ed by using 27 morphological traits. The dendrogram based on the morphologi­ cal attributes showed a relative separation of the different accessions based on fruits color and taste. Molecular analysis was performed for the accessions by using selected SSR and ISSR primers. The primers marked a high discriminating power (0.7 to 0.89). The dendrogram constructed on the base of UPGMA method showed 13 different groups. The clustering patterns indicated no loca­ tion nor local name specificity among mulberry accessions. The combination of SSR and ISSR primers was informative for estimating the extent of mulberry genetic diversity. It can be concluded that there is a high level of genetic diver­ sity within mulberry trees in Lebanon. These results will be useful for mulberry germplasm management in terms of biodiversity protection and as a valuable source of gene pool for crop improvement.


Introduction
Mulberry belongs to the genus Morus of the family Moraceae. It is a multipurpose tree with a significant ecological, nutri tional and economical high value. Mulberries are highly adaptable species in different soil and climatic conditions. They are generally quite tolerant to drought, pollution and poor soil. Therefore, they can be found in a wide area of tropical, subtropical, and temperate zones in Asia, Europe, North America, South America, and Africa (Kafkas et al., 2008). The genus Morus regroups 24 species (Thabti et al., 2014). The most widespread species in the Mediterranean climate areas are: Morus alba with fruit colors ranging from white to dark red, Morus rubra with mainly red/purple fruits and Morus nigra with dark purple to black fruits (Gerasopouls and Stravroulakis, 1997).
Mulberry fruits have remarkable potential for pro viding various valuable industrial products of high economic value for human beings. They are used for direct fruit consumption (Morus alba, Morus indica, Morus nigra, and Morus laevigata). Most of mulberry species have distinct flavor with juicy and acidic char acteristics making them attractive for use in the pro cessing industry for products such as fruit juice, ice cream, jelly, and jam (Ercisli and Orhan, 2007). Interest in mulberry has increased considerably over the last 20 years as a healthy fruit. Morus species have great antioxidant potential due to their high content in phenolic compounds including flavonoids, anthocyanins, and carotenoids (Zhang and Ma, 2018). Mulberries present anticancer and antiinflam matory properties and show as well significant effect on many chronic diseases like diabetes (Nakamura et al., 2009;Kwon et al., 2015;Qian et al., 2015).
Mulberry is an economically important plant used for sericulture. It is the sole food plant for the domesticated silkworm, Bombyx mori (Zhao et al., 2009). The genus Morus, is cultivated extensively in East, Central and South Asia for silk production (Awasthi et al., 2004). Hence, mulberry is one of the most important components that decide the sustain ability of this industry (Liu et al., 2009).
At the turn of the century, Lebanon was known for its highquality silk industry. Bestowed with an ideal climate and a fertile soil, mulberries were plant ed everywhere in Lebanon and mulberry production flourished (Firro, 2009). The silk tradition in Lebanon is more than two thousand years old. It goes back to the period of the famous purple dye (Ourjouan) extracted from the Murex shell by the Phoenicians of Sidon and Tyre and used to produce imperial purple silk (Khater, 2009). In the 19 th century, silk industry constituted almost 80% of Lebanon's economy. By the early 20 th century, 70% to 80% of the cultivable land of the country mountainous regions (Mount Lebanon) became devoted to mulberry orchards. Due to the high demands in silk production, mulberry tree has an unsurpassed economic impact on rural com munities. After 1940's, when silk began to be import ed from the Far East, the sericulture industry declined sharply. Mulberry cultivation became mar ginalized. However, Lebanon still has very old rainfed mulberry trees which are the remnants of the abun dant orchards that were once shaping the landscape of many villages. Mulberry trees are found in differ ent Lebanese villages, mostly located at orchards periphery or in small gardens.
In Lebanon, mulberry genotypes are very diverse, as they were sometimes obtained in the past from seeds or from cuttings. This process has led to a great number of landraces adapted to different conditions and different uses throughout the country. In Lebanon, there are many local traditional accessions but no named cultivars. Mulberries are distinguished and denominated according to the fruit color: "Abyad" (white mulberry), "Mwachah" (purple mul berry), "Shami" and "Aswad" (black mulberries).
Mulberry genetic diversity is progressively being lost in farmers' fields and in nature. The threat results from the interaction of several factors and is processing at an alarming rate. The most crucial fac tors are urbanization, climatic changes, out breaks of new diseases and pests, and the frequent occurrence of natural calamities. Little information is available about the genetic diversity of Lebanese mulberries. To protect mulberry in Lebanon, a marginalized species, conservation programs should be initiated. In this study, we have collected local mulberry acces sions from different geographical regions of Lebanon and assessed their genetic diversity by using agro morphological traits as well as molecular markers (SSR and ISSR).

Field survey
Samples of fruits for morphological and chemical analyses were collected from local trees of mulberry Morus from 21 sites covering different Lebanese regions (the North plain, Bekaa plain, Mount Lebanon, the South). These sites are subjected to dif ferent climatic conditions (precipitation, tempera ture) and agricultural practices. They are situated at an altitude between 30 and 1620 m, a latitude rang ing from N33° 16' 166" to N34° 21 '51.5" and a longi tude between E36°10' 849" and E35° 01' 38.7" (Fig.  1). The number of individual trees sampled per site (population) ranged from two to sixteen cultivars. In total, 70 accessions of mulberry were studied. Collected samples consisted of mature fruits (approx imately 500 g) and vegetative materials (young leaves, mature leaves and branches). The studied accessions included 'Abyad' (white mulberry), 'Mwachah' (purple mulberry), 'Shami' and 'Aswad' (black mulberries).

Morphological and chemical characterizations
The characterization of the vegetative materials and the fruits was based on descriptors for investiga tion of mulberry germplasm's morphology produced by Agriculture and Consumer Protection FAO (Sohn, 2003). Thus 27 morphological characters were stud ied for the mulberry accessions. These studied traits included 13 qualitative characters (for the leaf: shape, margin, base, apex, surface, color, glossiness, phytotaxis, bud shape and color; for the fruit parts: shape, color, taste, seed color) and 14 quantitative characters (for the vegetative parts: leaf length, width and thickness, petiole length, bud length, internode length; for the fruit parts: fruit length, diameter and weight, peduncle length, juice percent age (volume of the juice*100/weight of the fruits), sugar quantity (using refractometer), pH and acidity (by titration reaction).

Molecular characterization
DNA extraction. Genomic DNA was extracted from mulberry young fresh leaves using cetyl trimethyl ammonium bromide (CTAB) procedure described by FAO/IAEA (2007). The DNA quantity and quality was visually quantified using the agarose gel elec trophoresis method as described by Maniatis et al. (1982). DNA samples were stored at 20°C.

PCR amplification of the DNA with SSR primers.
Microsatellite polymorphisms were identified using three SSR primers (primers: MulSTR1, MulSTR2 and MulSTR3) (Tikader et al., 2009). Microsatellite ampli fication reactions were performed in a final volume of 25 µL in the presence of 2.5 µl of buffer, 200 µM of each dNTP, 0.4 µM of each primer pair, 1 unit (U) of Taq DNA polymerase, 50 ng template DNA, and 2 mM MgCl 2 . The amplification reaction consisted of an initial denaturation step at 94°C for 4 min, followed by 45 cycles of 1 min denaturation at 94°C, 65 sec annealing at 50°C, 90 sec extension at 72°C with a final extension of 72°C for 10 min using thermal cycles. The PCR amplification products were separat ed on a 6% denatured polyacrylamide gel and visual ized by silver staining.

Data analysis
For qualitative traits, scores were attributed according to FAO mulberry descriptors. A phenotypic diversity index, hsj (Shanon index) (Magurran, 1988) was calculated for each site to describe the pheno typic diversity of mulberry. The following formula was used for calculating hsj for each trait with n cate gories hsj=Ʃ P i LnP i where p i is the relative frequency in the ith category for the jth trait. The average diver sity (H) over k traits of each site was estimated as: H=Ʃ hsj/k. Traits evaluation was performed by using the Principal Component Analysis (PCA). The relation ships between mulberry leaves and fruits based on their quantitative and qualitative traits were studied using Hierarchical Cluster Analyses executed using Euclidean Distance following the Ward's method implemented in PAST software (Hammer et al., 2001).
To assess the information given by SSR and ISSR markers, the following parameters were calculated: number of alleles per locus, percentage of observed heterozygosity (Ho), expected heterozygosity (He= 1 Σpi 2 , where pi is the frequency of the i th allele) and the power of discrimination (PD = 1 Σgi 2 , where gi is the frequency of the i th genotype). Genetic distances were calculated according to Jaccard (1908). Trees were produced by clustering the data with the unweighted pairgroup method (UPGMA) with SAHN clustering and tree programs of PAST software (Saporta, 1990).

Morphological analysis
Leaves morphological characterization. Mulberry trees tend to have short trunks with large, low, spreading limbs. Leaves were alternately arranged and simple. The majority of cultivars had a cordate leaf shape, except the leaves of two accessions were reniform ('Abyad' and 'Shami') and two other were cordate to oval ('Abyad' and 'Mwashah'). All leaves had dentate margins. They presented mainly a cor date base and an acute apex. Leaf surface of 70% of the accessions was slightly rough. Only nine acces sions of 'Shami' presented rough surfaces. Leaves generally presented an average length between 6.83 and 18.9 cm and width between 3.94 and 17.02 cm. Petiole length average was between 0.72 and 5.07 cm. Leaves of black mulberry acces sions 'Shami' (0.02 and 0.03 cm) were thicker than those of white mulberries (0.01 cm).
Fruits morphological characterization. For the 70 accessions, the pomological characteristics investi gated showed a great diversity. Concerning the fruit shape, 35.7% of the mulberry fruits had oblong shape, 27.1% were round, 24.2% were reniform, and 13% were oval. For the local variety 'Mwashah', near ly half of the accessions had oblong shape while the majority of 'Shami' had round one. White mulberry 'Abyad' presented mainly oblong and reniform shape.
Fruit color of mulberry accessions was diverse: 'Abyad' accessions were white and 'Mwachah' acces sions were violet. The fruit color of 'Shami' acces sions were darker and varied between redpurple to black or black. Seed color varied between light yellow and yellowbrown; 'Mwashah' fruits had mainly light yellow seeds and 'Shami' presented yellowbrown seeds.

Morphological characterization PCA
The characterization of the collected mulberry accessions using different morphological characters showed high level of variation among the accessions. The Principal Component Analysis (PCA) revealed that the first 3 components explained 37% of the total variation, based on the 27 morphological char acters ( Table 2). The first component represented 18 % of the total variation and included fruit and leaves characteristics. It comprised fruit length, color, taste, pH, acidity and peduncle length, besides to the peti ole length, glossiness and thickness of leaves. The second component represented 10% of the total vari ation and is mainly influenced by leaf width. The third component was characterized by a percentage of variation of 9% and is dominated by the bud length character.

Classification of accessions based on morphological attributes
The accessions could be separated into groups based on the 11 most discriminant traits. The hierar chical cluster analysis classified mulberry accessions in 6 groups at 6 similarity of Euclidean distance (Fig.  2, Table 3).
'Shami' accessions were classified separately into 3 main groups (G1, G4 and G5). The first group G1 included 'Shami' and 2 accessions of 'Aswad'. G1, G4 and G5 accessions were characterized by a sour fruit taste and a dark blackpurple or blackred fruit color. Fruits of G4 and G5 presented significantly the lowest pH mean values (3.78 and 3.85 respectively). G5 fruits presented the lowest sugar content (9.62 °Brix). Accessions of G4 and G5 had the shortest fruit length (2.32 cm and 2.45 cm respectively) and the shortest peduncle (0.24 cm and 0.46 cm respective ly). Regarding leaf characteristics, G4 and G5 acces sions were characterized by low mean value of leaf length, while G1 were characterized by a significantly high leaf length (17 cm). G1, G4 and G5 accessions had also the thickest limb (0.02 0.03cm) and the shortest peduncle (1.4 1.48 cm).
The group G2 consisted of 12 accessions of 'Abyad' and one of 'Mwashah'. These accessions were characterized by a white fruit color and a sweet taste. The group G3 consisted of 19 accessions of 'Mwashah' and 2 of 'Shami'. They were characterized by purple fruits. They presented the highest value of fruit length (3 cm) and medium values for pH and Different letters were significantly different at the 0.05 level (Duncan's Multiple Range Test). sugar content. The group 6 regrouped 5 accessions of 'Mwashah', 10 of 'Abyad' and one of 'Aswad'. These accessions were characterized by their sweet taste (high sugar content and high pH). Groups 2, 3 and 6 presented a medium leaf thickness.
Molecular characterization ISSR analysis. The molecular analysis of mulberry accessions presented a high variability. The ISSR markers showed distinct polymorphism between the different mulberry accessions, only primers UBC826 and UBC864 showed no amplification. A total of 18 polymorphic bands were detected across the 70 accessions of mulberry through the use of four ISSR primers UBC807, UBC810, UBC827 and BI3. The size of amplified products ranged from 700 bp to 1600 bp. The number of scorable markers produced per primer ranged between 4 (UBC810, UBC827) and 5 (UBC807, BI3). This study showed that (AC), (GA) repeat primers generated excellent band profiles. Primers synthesized from (ATG) repeats failed to amplify.
The power of discrimination calculated for each primer (Table 4) enabled us to evaluate the genetic diversity of our locus. The calculated power of dis crimination PD values were between 0.75 and 0.89, showing that the studied loci are of high diversity.
These primers could be effectively used to study polymorphism between mulberry accessions. SSR analysis. Primer MulSTR1 showed no amplifi cation within our accessions. A total of 10 polymor phic bands were detected across the 70 accessions of mulberry through the use of two SSR primers (MulSTR2, MulSTR3). Upon using MulSTR2 SSR primer, the acrylamide gel showed three bands across the different accessions (192bp, 200bp, and 208bp), while MulSTR3 SSR primer generated 7 dif ferent bands ranging from 192bp to 275bp (Table 5). The results presented high polymorphism in all the amplified loci. In their assessment of mulberry geno types by SSR marker profile, Wani et al. (2013) used MulSTR2 and MulSTR3 primers which generated each 2 alleles among the 17 mulberry genotypes tested. Our results showed a higher number of polymorphic bands for a higher number of accessions. The power of discrimination was relatively high for each primer, PD>0.5. MulSTR3 presented higher polymorphism than MulSTR2, with a PD of 0.8 and an expected het erozygosity of 0.713. These two primers and especial ly MulSTR3 could be effectively used in genetic diver sity studies of mulberry.

Discussion and Conclusions
The conservation of the genetic variability of the ancient Lebanese mulberry trees is of utmost impor tance for germplasm preservation and for future breeding programs. Lebanese mulberries germplasm has not been assessed yet. This study is the first genetic diversity assessment of the Lebanese mulber ry germplasm using a set of morphological traits and genetic markers. Our inventories recensed accessions of four vernacular names 'Abyad', 'Mwachah', 'Shami' and 'Aswad' across different Lebanese regions. This shows that a limited number of tradi tional varieties was cultivated since decades, howev er morphological and molecular characterization of these 70 accessions revealed high diversity of this germplasm collection.
The results of morphological characterization revealed a high level of variation among mulberry characters. Among the 27 descriptors studied, 11 specific characters of fruits (Fruit taste, fruit length, fruit color, pH, titrable acidity and peduncle length) and leaves (petiole length, leaf thickness, leaf glossi ness, leaf width and bud length) revealed to be the most discriminating characters. The first component of the PCA was dominated by the fruit characteris tics. A broad morphological diversity of the fruit was reported for mulberry germplasm (Yilmaz et al., 2012;Peris et al., 2014;Aljane and Sdiri, 2016;Krishna et al., 2020). In our study, fruits exhibited dis tinct variations. Fruits shapes were diverse. Fruit color, titrable acidity, sugar content, juice yield and pH content were the most discriminating characters to differentiate mulberry accessions. Similar results were reported and significant differences were observed between the fruit characteristics (Yilmaz et al., 2012;Peris et al., 2014;Aljane and Sdiri, 2016;Krishna et al., 2020). Fruit color is a desirable charac ter for commercial acceptance of a variety. Fruit color of our accessions varied from white, red, purple to black. The percentage of juice yields were within the limits of Yilmaz et al. (2012) study (between 39% and 72%). All black mulberries had the highest fruit juice yield ratio, the highest acidity values and the lowest sugar content. This is consistent with previous researches (Ozdemir and Topuz, 1998;Gunes and Cekic, 2004;Aljane and Sdiri, 2016). Therefore, black mulberries are preferred for processing into juice. Black colored mulberry species received recently a great importance due to higher contents of phenolic compounds and to their delicious taste (Aljane and Sdiri, 2016). The dendrogram constructed on the base of the most discriminant morphological characters divided the accessions into 6 distinct groups. The evaluation of the relationship among accessions reduced their differentiation to fruit color and taste. A nonnegligi ble variability of other traits influenced the grouping involving the length of the fruit, leaf and peduncle. The accessions grouping was marginally correlated to the accessions vernacular names with many excep tions. The dendrogram revealed that accessions with in each cluster belonged to different regions suggest ing that there was no clear relationship between accessions and geographical diversity. This is the case of the group G4 that included 'Shami' accessions growing in North Lebanon (Kfarchakhna) and in South Lebanon (Sour). Such results have been report ed in different crops by several studies, e.g. on chest nut (Marinoni et al., 2013), almond (Chalak et al., 2007Halasz et al., 2019) and olives (Chehade et al., 2015). This variability could be attributed to the free exchange of planting material between different Lebanese villages and emphasizes the adaptability of mulberry to different ecological conditions.
In this study, we evaluated the genetic diversity and the relationships among the collected mulberry accessions using SSR and ISSR markers. The results showed high polymorphism in all the amplified loci. The power of discrimination values was high showing that the studied loci are of high diversity. The observed SSR markers heterozygosity were high. Earlier studies using amplified fragment length poly morphism (Sharma et al., 2000), ISSR (Awasthi et al., 2004), andRAPD (Xiang et al., 1995;Feng et al., 1996;Zhao and Pan, 2000;Esha and Shirish, 2001) also showed a large genetic variation among differ ent mulberry genotypes. Such a high level of poly morphism reflects the outcrossing nature of the species. In this work, the ISSR profiles generated by (AC) and (GA) repeat anchored primers showed that these repeats are abundant in our accessions. Vijayan and Chatterjee (2003) observed amplification of (AC) rich repeat based ISSR primers. Awasthi et al. (2004) concluded that (CA)/(TG) repeats are abun dant in Morus genome.
Cluster analysis of SSR and ISSR data using UPGMA revealed high genetic distances between the studied accessions. Five groups were constructed by one single accession. The other clusters regrouped accessions of 'Mwashah', 'Shami' and 'Abyad' within each group. The distanced genetic relationships among mulberry accessions are in consistence with their high heterozygosity due to their out breeding reproductive system (Dandin, 1998). Accessions from different varieties and from different sites were grouped together. The molecular results emphasized that genetic diversity among mulberry accessions is not influenced by their geographical ori gin nor by their local names. This finding is in agree ment with other researchers who studied genetic diversity using SSR markers on different crops, almonds (Distefano et al., 2013), mung bean (Wang et al., 2018) and torch Ginger (Ismail et al., 2019). The analysis of the genetic parameters showed the high diversity of mulberry in Lebanon.
The comparison between morphological and mol ecular diversity indicated that morphological descrip tors provide different information than the molecular one. In comparison with other works in woody species there were also no correlation. For example, two 'Shami' accessions (code B24 and HAS92) were in the same group in the morphological dendrogram however they belong to different groups in the mole cular one. One 'Aswad' (black mulberry, code B6) accession and another 'Abyad' (white mulberry, code B7) accession were in the same group in the molecu lar dendrogram but they were not in the morphologi cal one. It is probably that our markers sampled mainly a nonadaptive diversity.
The results of this study revealed a large morpho logical diversity and a high genetic variation among the Lebanese mulberry accessions. The combination of SSR and ISSR primers was informative for estimat ing the extent of mulberry genetic diversity. Morphological and molecular clusters have distin guished different lines of mulberry which may help in the selection of the most diverse profile. This germplasm would enhance the local gene pool and expand genetic variation for mulberry breeding pro gram in the future.