Impaginato 113 Adv. Hort. Sci., 2018 32(1): 113-135 DOI: 10.13128/ahs-21330 Bio-techniques for improvement of qualitative and quantitative traits in walnut (Juglans regia) U.N. Shah 1, J.I. Mir 2, N. Ahmed 3, A. Zaid 4, S. Jan 3, K.M. Fazili 1, S.H. Wani 5 (*) 1 Department of Biotechnology, University of Kashmir, Srinagar, Jammu and Kashmir, India. 2 Central Institute of Temperate Horticulture (ICAR), Srinagar, Jammu and Kashmir, India. 3 Sher-i-Kashmir University of Agricultural Science and Technology, Jammu and Kashmir, India. 4 Plant Physiology and Biochemistry Laboratory, Department of Botany, Faculty of Life Sciences, Aligarh Muslim University, 202002 Aligarh, India. 5 MRCFC, Khudwani, Sher-i-Kashmir University of Agricultural Science and Technology, Jammu and Kashmir, India. Key words: antioxidants, molecular markers, nutraceuticals, polyphenols, phy- tochemicals, walnut. Abstract: Walnut, Juglans regia (L.) is an economically significant plant for its immense nutritive and economic value. The breeding character of walnut has lent it a wide diversity in genetic characteristics. The principal vegetative and common traditional agronomic traits together with biochemical characteriza- tion i.e., karyotyping and isoenzyme expression have been the early research methods. However, these techniques are time-consuming and susceptible to the environmental variations. Literature is meager in the distribution, applied applications in general and the use of agriculture biotechnology in particular in case of walnut plants. The bio-techniques like molecular markers are adequate in number and there is little or no diversity in the method employed for research on walnuts. Despite basic research method, the organization of infor- mation, its retrieval and presentation structures, form elaboration experienced immense advancement via molecular markers such as RFLP, ISSR, RAPD AFLP, SSR and SNP. This appraisal in its first part provides detailed information regarding the present scenario of data on biogeographical distribution, health benefits of walnut worldwide and current applications in the agroforestry man- agement, biochemical evaluations and applied uses of a walnut tree which is relevant for both basic and applied research. The review in its second part sheds light on the application of sophisticated agricultural biotechnology tech- niques such as use of molecular markers to evaluate, realize the full potential of walnut for increasing its quality, quantity and for its sustainable production which cannot be obtained through usual breeding techniques to meet the demands of a projected world population. (*) Corresponding author: shabirhussainwani@gmail.com Citation: SHAH U.N., MIR J.I., AHMED N., ZAID A., JAN S., FAZILI K.M., WANI S.H., 2018 - Bio-techniques for improvement of qualitative and quantitative traits in walnut (Juglans regia). - Adv. Hort. Sci., 32(1): 113-135 Copyright: © 2018 Shah U.N., Mir J.I., Ahmed N., Zaid A., Jan S., Fazili K.M., Wani S.H. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 26 September 2017 Accepted for publication 27 October 2017 AHS Advances in Horticultural Science Review paper Adv. Hort. Sci., 2018 32(1): 113-135 114 1. Origin and history of walnut (Juglans regia L.) Walnut Juglans regia (L.) is commonly recognized as “Jupiter’s royal acorn”. English walnut is common- ly known as “Persian walnut” as it originated from ancient Persia and was adorned as for royalty. Juglans regia (L.) species is extensively cultivated in the temperate zones of the world for its best quality nuts and procurement of commercial timber (McGranahan and Leslie, 1991, 2009). Lateral fruit bearing walnut exhibit more genetic variation partic- ularly among familial varieties. In ancient times, the recognition of walnut was spread vigorously through caravans via sea trade. Greek and Romans have lately acquired trend towards walnut cultivation to avert the speckled walnut growing along the coastal areas. 2. Botanical description of walnut Plant morphology Juglans regia (L.) is a deciduous perennial tree belonging to the family Juglandaceae and grown chiefly for its edible seeds. Walnut tree is predomi- nantly an extensive canopy-dweller decurrent tree with trunk size ranging from 1.5-2 meters in diame- ter, 25-35 m in height and its survival rate is about 200 years. Walnut bears smooth bark with olive brownish color adapted to moderate light conditions. Leaves are 30-40 cm long, compound, alternately arranged and odd-pinnate. Male flowers are pro- duced in drooping catkins and the female flowers, in clusters of two to five, are terminal, which ripens in the autumn into a fruit with a semi-fleshy husk and a brown nut. The kernel of the nut is protected by a corrugated woody shell. Flowering, pollination and fruiting Juglans are monoecious species, having male and female reproductive organs on separate flowers on the same tree. Juglans regia is self-fertile, heteroga- mous and it can be protandrous or protogynous depending on cultivar. The characteristic inflores- cence is catkin bearing about 2-3 pistillate flowers born terminally or laterally. Walnut is self-fertile however occasionally it requires different cultivar due to dichogamy. Majority of walnuts are protan- drous and anemophilous. Nuts are borne individually or in groups enumerating 2-3 on shoot tips. Walnut is properly shielded by a fleshy covering which cracks at maturity. Walnut shape is variable from ovoid to round attaining a maximum diameter of 2 meters enclosing two kernels divided by extremely delicate covering expanding from inner cover of the shell. Taxonomical classification Walnut is a member of Juglandaceae family and includes approximately 60 species, among which 21 species are classified under genus Juglans (Table 1). Different varieties of walnut and their commercial uses are given in Table 2. All walnut species are edi- ble and English walnut is easily crackable and enor- mous in size. Different sections and species of walnut Genus Juglans is classified into four types: Juglans sect. Cardiocaryon, Juglans sect. Juglans, Juglans sect. Rhysocaryon, and Juglans sect. Trachycaryon. Juglans sect. Cardiocaryon. This variety originated from the northeast Asia. This walnut variety bears enormous leaves containing 15 leaflets. The wood is commercially soft, and nuts are thick shelled borne in a raceme inflorescence, such as, for example, J. mandshurica and J. ailantifolia. Juglans sect. Juglans. Originated from the south- east Europe to central Asia. This variety bears large leaves with 9 broad hairless leaflets having entire margins. The wood is commercially hard, for exam- ple, J. regia (L.) and J. sigillata. Juglans sect. Rhysocaryon. (black walnuts) Originated from the North America and the South America. This variety bears enormous leaves with 20 Table 1 - Taxonomic classification of walnut Kingdom Plantae Phylum Tracheophyta Class Magnoliopsida Order Fagales Family Juglandaceae Subfamily Juglandoideae Tribe Juglandeae Subtribe Juglandinae Genus Juglans Species ● J. regia Linnaeus ● Juglans ailanthifolia Carr. ● Juglans ailantifolia Carriere ● Juglans bixbyi Rehd. ● Juglans boliviana (C. DC.) Dode ● Juglans californica S. Wats. ● Juglans cinerea Linnaeus ● Juglans hindsii (Jepson) Jepson ex R. E. Sm. Shah et al. - Bio-techniques for improvement of qualitative and quantitative traits in Walnut. A review 115 Table 2 - Different varieties of walnut and their commercial uses No. Species Description Commercial uses 1 English/Persian Walnut (Juglans regia) Native to the region of California, Oregon, Himalayas, Europe and China. Most ubiquitous cultivated in foothills having high water table. Characterized by a relatively thin, gnarled shell enclosing a smooth, large, ivory-colored nut and rich flavor. As an effective nutraceutical with a high nutritive value. Procurement of high quality timber in the furniture industry for high-end flooring, guitars, furniture, veneers, knobs and handles as well as gunstocks 2 Eastern Black Walnut (Juglans nigra) Native to eastern North America. Characterized by a thick, hard shell with sharp, jagged edges and a darker color. Black nuts are known for their pungent aroma and robust flavor. Used in timber industry as well as nut production. The hard- black walnut shell is also used commercially in abrasive clea- ning, a filtering agent in scrubbers in smoke stacks, cleaning jet engines, cosmetics, and oil well drilling and water filtration 3 White/ Butternut (Juglans cine- rea) Native to the eastern United States and southeast Canada. Characterized by slow-growing species, and rarely lives longer than 75 years. Bark is light grey in colour and the whole leaf is downy-pubescent, and a somewhat brighter, yellower green than many other tree leaves. Leaves are alternate and compound and have odd number of leaflets with a terminal leaflet Used in lumber industry to make furniture, and is a favorite of woodcarvers. It is used to dye fabrics. Used as a medicine owing to its cathartic properties 4 Japanese Walnut/ Heartnut (J. ailantifolia) Native to Japan and Sakhalin. Characterized by light grey bark, pinnate and brighter yellower green leaves, nuts produced in bunches of 4-10 and are of spherical shape The edible nuts have an oily texture. The husks are also used to make a yellowish dye 5 Manchurian/ Chinese Walnut (J.mandshurica) Native to the eastern Asiatic region (China, Russia and Korea). Characterized by odd, alternate, pinnate, long and broad leaves. Shell is thick and the kernels are edible but small and difficult to extract. Also contain less quantities of allelopathic compounds like Juglone The timber is in use but is less valuable as compared to English or Black walnut. Cultivated as an ornamental in colder tempe- rate regions 6 Iron Walnut (Juglans sigillata) Native to the Yunnan, Guizhou, Sichuan and Xizang in China. Characterized by oval-shaped nuts with bumps and ridges. Cultivated for its edible nuts Used as ornamental plant in gardens and parks 7 California Walnut (Juglans californica) Endemic to California and generally found in southern California Coast Ranges, Transverse Ranges, and Peninsular Ranges, and the Central Valley. Characterized by a large shrub or a small tree. It has a small hard nut in a shallowly grooved, thick shell that is difficult to remove. It has a better flavour than Juglans nigra Raw as well as cooked seeds are used in pies, cakes, biscuits, confections etc. An attractive wood, but the frequent bran- ching pattern of the trunk limits the use of this wood commer- cially. Also has a medicinal value 8 Brazilian/ Argentine walnut (J. australis) Native to Argentina and Bolivia. Characterized by dense, hard and strong wood. Its more frost resistant than J. regia Nutritive value. The immature fruits are pickled whole for human consumption. The mature nuts are also eaten. The concentrated extract of the husk is also used as a vermifuge 9 Northern California wal- nut (Juglans hindsii) Endemic to Northern California. Characterized by smooth, brown, thick shell, that contains a small edible nutmeat Commercially important as a rootstock for orchard stock of Juglans regia. Also used as an ornamental tree in wildlife gardens, and for habitat gardens, natural landscaping projects. Its wood commonly called claro walnut is used in the lumber industry 10 West Indian walnut (West Indian walnut) Not native to Jamaica but found in Cuba, the Dominican Republic, Haiti, and Puerto Rico. Characterized by a drupe 2-3 cms long with a black husk and a seed, which is an edible walnut meat, inside. It is listed as an endangered species in the Endangered Species Act of the United States Used in timber industry. The attractive wood is similar to that of the black walnut 11 Cedro negro (Juglans olan- chana) Native to Costa Rica, Guatemala, El Salvador, Honduras, Mexico and Nicaragua. Commonly called cedro negro. Characterized by long branches bearing twigs tipped with 40-50 cm long, glabrous, pinnately compound leaves, darker on the top than on the bottom. The base of the trunk sometimes has buttresses Used in the timber industry for light construction, cabinet- making, parquet floors, luxurious furniture, turnery, musical instruments, and veneer. The husk is used to dye leather 12 Colombian walnut (Juglans neotropica) Native to Colombia, Ecuador, and Peru. Characterized by slow- growing tree up to 40 mts height with grooved, red-brown bark and an oval-shaped canopy. Leaves are compound with a serrated border Nuts are used as food. Used in timber industry because the hard, durable wood is highly prized in cabinetry, flooring, veneers, utensils, and other forms of decoration Adv. Hort. Sci., 2018 32(2): 113-135 116 leaflets, serrate margins. The wood is commercially very hard, such as, J. californica, J. hindsii, J. australis, J. nigra, and J. olanchana. Juglans sect. Trachycaryon. Originated from the Eastern North America. This variety bears wide leaves containing 20 leaflets having serrate margins. The wood is commercially soft and the fruits are borne in clusters of two to three. The nuts have a thick and rough shell such as J. cinerea (L.). 3. World walnut production Walnut grows abundantly in temperate areas of the world and cultivated commercially in 48 coun- tries on an area of 1.6 million acres. The worldwide production of walnuts has reached maximum partic- ularly in countries of Asia. Global walnut production for the year 2015-16 rose 155,000 tons from the pre- vious year to 2.1 million tons, with China and the United States accounting for over 75 percent of total production (USDA, 2016). China is the world’s chief producer of walnut and accounts for 51.49% of world’s walnut production followed by the USA (26.86%), European Union (6.02%), Ukraine, Chile, Turkey and India contribute 5.25%, 3.09%, 3.09%, 1.96%, respectively to the world’s walnut production (USDA, 2016). Global walnut production for the year 2016-17 is nearly unchanged from the October 2016 record forecast of 2.1 million tons in-shell basis, with China and the United States accounting for nearly 80 percent of total production (USDA, 2016). Figure 1 gives the schematic representation of year wise pro- duction of walnuts across the world. 4. Status of walnut in India Walnuts are commonly called “Akhrot” in India and they are grown in the northwestern Himalayan belt, expanding up to Sikkim and Darjeeling. But the commercial farming of walnut is limited to the states of Arunachal Pradesh, Himachal Pradesh, Uttarakhand and Jammu and Kashmir. The area and production of walnut in India for the year 2014-15 were recorded to be 125,000 ha and 206,000 tons respectively (Government of India, 2015). India has exported 3,291.71 MT of walnuts worth of Rupees. 117.92 crores during the year 2015-16 (APEDA, 2016). Figure 2 gives the schematic representation year wise area versus production of walnut in India. The major walnut importing countries from India are Vietnam Social Republic (15%) followed by Egypt Arab Republic (11%), Netherland (11%), United Kingdom (9%), Spain (8%), United States (7%), Germany (6%), France (4%), Thailand (3%), Australia (3%) and others (23%) (Ministry of Agriculture, 2015) (Fig. 3). Jammu and Kashmir is the main center for com- mercial walnut production in India and contributes pretty nearly 98% of the country’s output and the Fig. 1 - Schematic representation of walnut year wise produc- tion across the world. Fig. 2 - Shows the schematic representation of walnut produc- tion during three consecutive years in India. Fig. 3 - Showing principal countries which imports walnuts from India. Shah et al. - Bio-techniques for improvement of qualitative and quantitative traits in Walnut. A review 117 state has been declared as the “Agri. Export zone for Walnuts” (Isher et al., 2016). Most walnuts that are produced for export by India are principally produced in this state. According to the United Nations Development Programme Special Unit for South- South Cooperation (UNDP SU/SSC), 63,000 hectares of Jammu and Kashmir state is under walnut cultiva- tion. These plantings produce around 60,000 tones of walnuts worth an estimated 25 million Rupees. 5. Walnut as phytochemical Walnut is popularly known for its abundant phyto- chemicals for instance phenolics, fatty acids, mela- tonin and serotonin (Christopoulos and Tsantili, 2015). In addition to kernels, green shells, walnut husk, seeds, bark and leaves are utilized in the cos- metic and pharmaceutical sector (Negi et al., 2011; Fernández-Agulló et al., 2013; Vinson and Cai, 2012). Table 3 gives a description of walnut and its phyto- constituents in diverse walnut varieties. The level of phenolics in walnut depends on diverse ecological factors, genotype, type of cultivar, geographical site, climatic parameters and developmental stages (Solar et al., 2006; Amaral et al., 2008). Toivonen and Hodges (2011) illustrated tissue and age specific vari- ation in phenolic acids. Walnut polyphenols are potential candidates which work against in vitro plas- ma and low-density lipoprotein LDL oxidation. Regueiro et al. (2014) reported several individual phenolic compounds in walnut kernels and charac- terized approximately thirty-seven compounds. Colaric et al. (2005) demonstrated a greater amount No. Walnut name Part used Phytochemical extracted Reference 1 Persian walnut Kernels Phenolic acids (gallic acid, ellagic acid, syringic acid, chlorogenic acid, p-coumaric acid) and flavanols (catechin, epicatechin, gallocatechin, procya- nidin B2, epigallocatechin, epicatechin gallate) Figueroa et al., 2016 2 Persian walnut Kernels Ellagic acid Fukuda et al., 2004; Christopoulos and Tsantili, 2012 3 Heartnut Juglans ailanthifolia var. cordiformis) and Persian walnut (Juglans regia L.) Kernels Ellagic acid Li et al., 2006 4 Persian Walnut Leaves Phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salycilic, ellagic and trans-cinnamic) flavonoids (catechin, epicatechin, rutin, myricetin and quercetin) and juglone Nour et al., 2012 5 Persian Walnut Bark Gallic acid, chlorogenic acid, catechine hydrate;resorcinol; caffeic acid, rutin trihydrate, syringic acid, vanillicacid,p-coumaric acid,quercitine dihydrate, naphto-resorcinol, trans-cinnamic acid etc Noumi et al., 2012 6 Persian Walnut Fruit Phenolic acids (gallic, vanillic, chlorogenic, caffeic, syringic, p-coumaric, ferulic, sinapic, salicylic, ellagic and trans-cinnamic acid) flavonoids (catechin, epicatechin, rutin, myricetin and quercetin) and juglone. Cosmulescu et al., 2014 7 Persian Walnut Walnut husks Chlorogenic acid, caffeic acid, ferulic acid, sinapic acid, gallic acid, ellagic acid, protocatechuic acid, syringic acid, vanillic acid, catechin, epicatechin, myricetin, and juglone. Stampar et al. 2006 8 Persian Walnut Kernels and pellicle Chlorogenic, caffeic, p-coumaric, ferulic, sinapic, ellagic, and syringic acid. Syringaldehyde and juglone Colaric et al., 2005 9 Persian Walnut Leaves and husks Chlorogenic acid, ferulic acid, gallic acid and trans-cinnamic acid Chrzanowski et al., 2011 10 Persian Walnut Leaves Juglone Thakur, 2011; Babula et al., 2006 11 Persian Walnut Axillary shoots (microshoots) Phenolic acids (gallaicacid, chlorogenic acid, p-coumaric acid, syringic acid, vanillin) and flavanoids (quercetin, naphthoquinone) and juglone Cheniany et al., 2013 12 Persian Walnut Fruit Gallic acid, chlorogenic acid, sinapic acid, protocatechuic acid, catechin and juglone Jakopič et al., 2009 Table 3 - Description of walnut and its phytoconstituents in diverse walnut varieties Adv. Hort. Sci., 2018 32(1): 113-135 118 of phenolics in walnut skin than in kernels. Oliveira et al. (2008) confirmed protective properties of walnut husk exhibiting higher anti-microbial activity. Walnut kernels are known to be an immense resource of ellagitannins having higher anti-oxidant property (Shimoda et al., 2009). Amaral et al. (2008) con- firmed that presence of 96% of tannins as major causal agent for the medicinal potential of walnuts along with phenolics and flavanoids. Phenolic com- pounds are known to confer health benefits. Juglans regia L. leaves are good sources of flavonoids. Many types of research are mainly focused on the extraction/isolation and the antioxi- dant effect of flavonoids. Walnut flavonoids have not been extensively analyzed, however, English walnut leaf was subjected to vigorous chromatographic eval- uation (Pereira et al., 2007). Amaral et al. (2008) and Pereira et al. (2007) analysed quercetin 3-galacto- side, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside and two other partially iden- tified quercetin 3-pentoside and kaempferol 3-pento- side derivatives in walnut leaves along with myricetin as exclusive flavonoid in walnut husk (Lugasi et al., 2003; Stampar et al., 2006). Myricetin exhibit excep- tionally high anti-oxidant property along with the potential of imparting beneficial effects on bone heath thereby preventing bone resorption and ele- vating osteoblastic potential and bone formation (Hsu et al., 2007). Rutin (Quercetin-3-rutinoside) is another significant flavonoid in walnut leaves having elevated levels of anti-oxidant potential that prevent DNA oxidation (Nour et al., 2012). The previous studies on flavonoids present in Juglans regia (L.) leaves provided a conjectural source for an additional study on the organic decoc- tion from J. regia leaves. The promising results from the medicinal point of view were procured from flavonoids. Estimation and characterization of pheno- lic compounds in walnut leaves can be utilized as an important resource. Carvalho et al. (2010) reported phenolics and associated anti-oxidant characteristics of methanolic and petroleum ether extracts procured from walnut green shells and leaves. Seeds methano- lic extracts exhibited highest phenolic content approximately 116 mg gallic acid equivalent (GAE)/g of extract with EC50 of 0.143 mg/mL using DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity followed by leaf and green husk. Solvent polarity was directly correlated with the distinct disparity in the activity of samples as observed by the solubility of phenolics compounds in respective solvents. Hence, methanol solvent is preferred for phenolics extrac- tion (Do et al., 2014) while petroleum ether is non- polar normally employed for extraction of lesser polar constituents for instance plant pigments, sterols and fatty acids (Carvalho et al., 2010). Carvalho et al. (2010) reported maximum phenolics fraction (116.22±3.76 mg of GAE/g of seed extract followed by leaf 94.39±5.63 mg of GAE/g of extract) and green husk (50.18±2.69 mg of GAE/g of extract). Previous research reports have conformed methano- lic extracts as most efficient in radical scavenging activity. Al-Okbi et al. (2014) reported phenolics con- tent of 47.42 g GAE/100 g in ethanolic extract of wal- nut seed. Jalili and Sadeghzade (2012) illustrated the average value of 22.16±0.66 (mg/g) of phenolic con- tent in aqueous extracts in diverse walnut cultivars. Ghasemi et al. (2011) illustrated the phenolic content of 15.15 to 108.11 mg gallic acid equivalents g-1 of extract and flavonoid content ranging from 3.59 to 22.91 mg quercetin equivalents (QE)g-1 of extract. Rahimipanah et al. (2010) reported total flavonoids and phenolics in walnut green husks via aluminum nitrate and Folin-Ciocalteu colorimetric protocol and their content were 1.44±0.2 mg quercetin and 34.28±1.35mg gallic acid equivalent per gram of dry sample correspondingly. Kale et al. (2010) demon- strated flavonoid fraction of walnut bark extracts with maximum quercetin between 3.50 to 32.81 mg/g and phenolics content varied from 20.32 to 44.87 mg/g in the extract. Ethyl acetate extract of walnut showing maximum phenolics and flavonoid sustaining antioxidant potential walnut extracts and recommend their utilization for advanced research to identify secondary metabolites. 6. Phytopharmacology of walnut All the parts of walnut i.e., bark, leaves, flowers are exclusively used widely in Ayurveda, Unani, homeopathy and allopathic medicine system (Shah et al., 2014). Forino et al., 2016 reported conventional uses of walnut in health maintenance. Diverse activi- ties of walnut utilized are due to enormous phyto- constituents such as saponins, glycosides, alkaloids, and steroids (Muthaiyah et al., 2011). All parts of walnuts particularly leave and husk is extensively used in the pharmaceutical and cosmetic industries (Ribeiro et al., 2015). Table 4 gives the description of walnut as phytochemical and its activity against diverse pathogens. Shah et al. - Bio-techniques for improvement of qualitative and quantitative traits in Walnut. A review 119 Antimicrobial activity Antimicrobial potential of walnut especially bark (Farooqui et al., 2015) leaves (Pereira et al., 2007) and fruits (Pereira et al., 2008) have been extensively utilized against various infections. Studies have also demonstrated the antimicrobial activity of walnut products, particularly of bark, leaves, fruits and green husk of Juglan regia (L.) was subjected to hot and cold extraction and resulting solution exhibited high antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus epidermidis, Micrococcus luteus, Salmonella typhimurium, Enterococcus faecalis, Bacillus thuringiensis, Protomonas extroquens, and Proteus sp., as evaluated through agar streak and disc diffusion assays (Biyik, 2010; Deshpande et al., 2011). Antifungal activity Aqueous extracts of leaves and bark display anti- fungal activity against an extensive spectrum of fungi using disc diffusion, agar dilution, agar streak dilution and reddish assays. Pereira et al. (2008) demonstrat- ed the enormous anti-fungal potential of walnut extracts refluxed with petroleum ether (b.p. 40-60°C) against Candida albicans and Cryptococcus neofor- mans. Noumi et al. (2010) demonstrated antifungal properties of Juglans regia (L.) alongside oral Candida strains. Antiviral activity Zhai et al. (2007) confirmed inhibition of tobacco mosaic virus (TMV) by 95% ethanol and ethyl acetate leaves extract of J. regia. At a minimum concentra- tion of 1.5 µg/ml of methanolic extract of J. regia, there was distinct inhibition Sindhis (Mouhajir et al., 2001). Antioxidant activity Several research reports demonstrated antioxi- dant activity of ethyl acetate, butanol, ether and aqueous methanol extract of walnut kernels, husks and leaves as evaluated by DPPH radical scavenging assay and lipid oxidation inhibition by β-carotene linoleate (Oliveira et al., 2008; Pereira et al., 2008; Carvalho et al., 2010; Rahimipanah et al., 2010; Table 4 - Description of walnut as phytochemical and its activity against diverse pathogens Walnut Part used Target cell/ pathogen Activity Reference Juglans regia Bark Staphylococcus aureus, E. coli Antibacterial Farooqui et al., 2015 Juglans regia Bark Candida albicans, Candida glabrata, and Candida parapsilosis strains Antifungal Noumi et al., 2010 Juglans regia Kernels MCF-7 (estrogen receptor positive breast adenocarcinoma), KB (oral and mouth), HepG-2 (liver), Caco2 (colon), and WRL-68 (liver) Anti-proliferative Negi et al., 2011 Juglans regia Leaves Tobacco mosaic virus Antiviral Zhai et al., 2007 Juglans regia Leaves Sindbis virus herpessimplex (HSV), Sindbis (SINV), and Poliovirus Antiviral Mouhajir et al., 2001 Juglans regia Leaves 769-P renal and Caco-2 colon cancer cells Anti-proliferative Carvalho et al., 2010 Juglans regia Leaves A375 human melanoma cell Anti-proliferative Shah et al., 2015 Juglans regia Kernels COLO-205 Anti-proliferative Anjum et al., 2016 Juglans regia Walnut oil Herpes simplex (HSV), Parainfluenza (PI-3) Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumo- niae, Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus faecalis, Fungi: Candida albicans and Candida parapsilosis Antiviral, Antibacterial, Antifungal Orhan et al., 2011 Juglans regia Leaves Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumo- niae, Fungi: Candida albicans, Cryptococcus neoformans Antibacterial and antifungal Pereira et al., 2007 Juglans regia Husks Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Fungi: Candida albicans, Cryptococcus neoformans Antibacterial and antifungal Oliveira et al., 2008 Adv. Hort. Sci., 2018 32(1): 113-135 120 Qamar and Sultana, 2011). Consumption of walnuts and walnut skin diet by C57BL/6 mice showed decline oxidative stress as observed by a change in the enzymatic and non-enzy- matic system (Bulló et al., 2010). Furthermore, research report demonstrated ameliorating effect of walnut consumption on LDL oxidizing potential owing to their elevated levels of omega-6 PUFAs. Antidiabetic activity In Mediterranean and Asiatic countries, air-dried leaves of Juglans regia have been employed to treat diabetic symptoms (Hosseini et al., 2014), and the potential of walnut leaves to ameliorate hyper- glycemia in humans has been evaluated by both in vitro and in vivo. Pitschmann et al. (2014) reported inhibition of protein-tyrosine phosphatase 1B and no effect on peroxisome proliferator-activated recep- tors-gamma (PPARγ) in myocytes fed with methano- lic extracts from walnut leaves. This study confirmed walnut leaf extracts drastically decline levels of serum fasting HbA1c, blood glucose and insulin (Hosseini et al., 2014). Hence the study concluded hypoglycemic potential of walnut leaves wielding lowering effect on sugar levels in the liver as well as kidney. The mechanisms of action underlying the antihyperglycaemic effect of the walnut leaf extracts were investigated by in vitro studies. Inhibition of sig- nificant enzymes involved in sugar metabolisms like α-amylase (Rahimzadeh et al., 2014), PTP1B (Ahmad et al., 2012) and associated processes like glycation and oxidation reactions have been reported with walnut leaves (Pitschmann et al., 2014). Antihelmintic activity A wide range of anti-worm activities was also reported from bark extracts of walnut. Kale et al. (2011) reported antihelminthic activity against Eiciniafeotida using reference Albendazole. Methanolic, ethanolic and benzene extracts of Juglans regia (L.) exhibited inhibitory activity against annelids like Pheretimaposthuma using standard drug Piperazine citrate (Upadhyay et al., 2010). Anti-inflammatory activity Anti-inflammatory potential of walnut is compara- tively inadequate. Papoutsi et al. (2008) demonstrat- ed anti-inflammatory activity because of ellagic acid. Hepatoprotective activity Ameliorative effect of walnut polyphenols from kernel pellicle against a decline in glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase (GPT) was reported in mice model after a single oral administration (200 g/kg) (Shimoda et al., 2008). These results confirmed higher values of a hepatoprotective effect than curcumin. 7. Walnut and cancer Due to the increasing inclination towards plant based drugs, more vigorous initiatives have been taken to action to develop more advanced plant based drug formulations. Among the diverse phyto- chemicals procured from plants, polyphenols are known to be most active candidates against cancer (Kim et al., 2016). Singh and Sukhla (2015) demon- strated polyphenols as potent cancer inhibiting phy- toconstituents as revealed by inhibitory effects of walnut on cancer promotion and progression. Polyphenols inhibit cancer progression by hampering nuclear factorkappa B - (NF-kB) activation. NF-kB is a nuclear transcription factor exhibiting significant role in gene regulation implicated in inflammation and carcinogenesis. Upregulation of NF-kB is implicated in cell-cycle progression. Polyphenol suppresses NF- kB and activator protein-1 (AP-1), its inhibition is con- sidered as a potential step in chemoprevention. Polyphenols further induce inhibition in mitogen acti- vated protein kinases (MAPK), protein kinases (PK), of the growth-factor receptor (GFR) leading to cell cycle arrest, the onset of apoptosis and decline in angiogenesis (Bonfili et al., 2008). Mertens-Talcott et al. (2005) illustrated on the synergistic, additive, or antagonistic interface of polyphenolic compounds to counteract cancer growth. Shah et al. (2015) report- ed numerous chemical constituents possessing anti- cancer activity due to omega-3 fatty acids (Gerber, 2012), vitamin E (mainly the c-tocopherol form) (Albanes et al., 2014), phytosterols (Ramprasath and Awad, 2015), ellagic acid (Eskandari et al., 2016), gal- lic acid (Ho et al., 2013) and flavonoids namely quercetin (Firdous et al., 2014), carotenoids (Virtamo et al., 2014), and melatonin (Travis et al., 2014). Hardman (2014) reported 80% inhibition of breast cancer in mice following walnut consumption. Due to the presence of omega-3 fatty acids along with phy- tosterols in walnut breast cancer was declined remarkably. Phytosterols bind with estrogen recep- tors and subsequently decline breast cancer. Consumption of walnut diet declined a breast tumour by about 60% in transgenic mouse. Hardman (2014) confirmed presence of numerous complex constituents in walnut exhibiting additive or synergis- tic effect on cancer suppression. Further presence of Shah et al. - Bio-techniques for improvement of qualitative and quantitative traits in Walnut. A review 121 γ-tocopherol in walnut exhibit anti-cancer activities against prostate, colon, renal and lungs as demon- strated by anti-proliferative and anti-angiogenic mechanisms (Rafieian-Kopaie and Nasri, 2015). Walnut inhibits inflammation by inhibiting endothelin (Ma et al., 2010). Research on prostate cancer estab- lished higher levels of endothelin in men indicating the need for further research to decipher relation between walnut and prostate cancer. Reiter et al. (2013) investigated prostate cancer and a deter- mined decline in tumor and LNCaP (Lymph Node Carcinoma of the Prostate) xenograft growth. Negi et al. (2011) determined anti-proliferative activity of chloroform and ethyl acetate fractions of walnut in human cancer cell lines such as MCF-7 (Michigan Cancer Foundation-7) (estrogen receptor positive breast adenocarcinoma), KB (oral and mouth), HepG- 2 (liver), Caco2 (colon), and WRL-68 (liver) cancer cell lines. All the cell lines exhibited inhibition against kid- ney and colon cancer however for 769-P renal and Caco-2 colon cancer cells, walnut leaf revealed the elevated rate of anti-proliferative competence than walnut seeds and husk. The observations procured confirmed lack of relationship among total phenols and anti-proliferative activities signifying some class of compounds implicated in cancer inhibition (Carvalho et al., 2010). This study was further validat- ed by Tsoukas et al. (2015) who demonstrated a con- verse association between walnut intake and colon cancer incidence. Paur et al. (2010) reported dose dependent inhibition in lipopolysaccharide(LPS)- induced NF-κB activity in the monocytic cell line (U937-κB) by blending clove, oregano, thyme, walnut and coffee. This study confirmed the comparative efficacy of the combination of different extracts than individual plant constituent. Hence, walnut can be utilized as a natural source of anti-oxidants as well as chemo preventive candidate. Thakur (2011) demon- strated inhibition of carcinogenesis in rats by azoxymethane and can be utilized as chemo preven- tive agent against neoplasia. During recent years, juglone is established as effective cytotoxic agent and induce cell apoptosis via mitochondria dependent pathway via human lung cancer (A549) cells (Cenas et al., 2006), human leukemia (HL-60) cells (Xu et al., 2012 a), and human cervical carcinoma (HeLa) cells (Zhang et al., 2012). Ji et al. (2008) reported inhibi- tion of growth and induction of apoptosis of sarcoma 180 cells in vivo. Juglone, being an inhibitor of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is an impending remedial target for anti- cancer research. Role of Pin1 in relation to tamox- ifen-resistant breast cancer has been an important determinant factor in drug resistance. These observa- tions demonstrate Pin1 augment E2F-4 and Egr-1-dri- ven expression of LC-3, an autophagosome marker is over expressed in cancer cells than non-cancerous. Namgoong et al. (2010) further established juglone as potent inhibitor having a distinct declining effect on the TPA and induced overexpression of E2F-4 and Egr-1 transcription factors controlling LC-3 gene expression. 8. Walnut as a nutraceutical The term “nutraceutical” is used to illustrate food or food nutrient supplement that suggests a medical or health benefit by providing simple nutrition (Wong et al., 2015). Functional foods are known to be a rem- edy for chronic diseases (Luciano, 2014). Walnut has been used as functional food. They are important components of the Mediterranean diet (Ley et al., 2014). Nutritional profile of walnut is tabulated in Table 5. Not only the kernel of walnut but also green shells and bark have also been used in the cosmetic and pharmaceutical industries (Ribeiro et al., 2015; Adeel et al., 2017). Walnuts are chiefly consumed for their organoleptic and nutritional aspects since they are resources of important anti-oxidants and pre- serve food attributes (Martinez and Maestri, 2016). Walnut seeds are now established as major nutraceu- tical owing to their effects on coronary heart disease (Nasri et al., 2015; Schwingshackl et al., 2017). Walnuts possess numerous nutritional aspects including a maximum fraction of polyphenolics due to which they are incorporated into recommended dietary supply (Sanchez-Gonzalez et al., 2017). Christopoulos and Tsantili (2015) reported numerous phytochemicals in walnut for example phenolics, fatty acids, melatonin and serotonin. In addition to phytochemicals, various hydrolyzable and condensed tannins have been reported in walnut (Figueroa et al., 2016). High oil and protein content of kernel of Juglans regia (L.) (Juglandaceae) validates this fruit requisite for human nutrition. FAO has categorized walnut as a deliberate candidate for human nutrition and phytomedicine (Gandev, 2007). Most of the energy in walnuts generates from fats which account for 65% which makes them energy-rich and high- calorie food (Tapsell et al., 2009). Martinez and Maestri (2008) reported triacylglycerols, in which monounsaturated fatty acids (MUFAs) mainly oleic acid (18:1 n-9) and polyunsaturated FAs (PUFAs; Adv. Hort. Sci., 2018 32(1): 113-135 122 linoleic (18:2 n-6) and α-linolenic acids (18:3 n-3) in walnut oil are present in high amounts in all geno- types. Walnuts are a chief resource for omega-6 fatty acid called linoleic acid. They also contain a relatively high percentage of omega-3 fat called alpha-linolenic acid (ALA) contributing up to 8-14% of the total fat content. Deckelbaum and Torrejon (2012) reported walnut as the sole source of ALA which declines inflammation and augments blood profile. Muradoglu et al. (2010) and Martinez et al. (2010) reported average protein estimation of about 18.1% comprising of 70% of total seed protein along with lesser amounts of globulins (18%), albumins (7%) and prolamins (5%). Walnut proteins possess all essential amino acids requisite for humans. Amino acids exist in proper ratio in walnuts e.g., lysine/arginine ratio in walnut proteins is comparatively lower than in daily vegetable proteins. The lower lysine/arginine ratio declines atherosclerosis development (Martinez et al., 2010). In addition to macronutrients, walnuts are richest sources of vitamins and minerals, Potassium (K), phosphorus (P), magnesium (Mg) and iron (Fe) (Cosmulescu et al., 2009). Calcium (Ca), sodium (Na), zinc (Zn) and Copper (Cu) are present in moderate fractions (Siahnouri et al., 2013). Macro elements are necessary for proper development and growth of organisms (Jeszka-Skowron et al., 2016). Most of macroelements assist in enzymatic reaction involved in the general metabolism of the organism. Trace ele- ments promote heart health, maintains bone, nerve and immune system functions (Abdel-Aziz et al., 2016). Vitamins are indispensable organic substances and in addition to their role in important metabolic functions, they are also required for proper function- ing of hormone regulation. Anjum et al. (2016) con- firmed presence of ellagic acid, gallic acid, tocopherol (vitamin E), ellagitannins (tannins) and phytochemi- cals with potential antioxidant activity, for instance, melatonin. Walnut also comprises of elevated levels of α- tocopherol, a vitamin E, which prevents lipid oxidation process and decline risk of cancer and coronary heart disease (Jiang, 2014). Health effects of walnut (Table 6) Cardiovascular benefits. Nijike et al. (2015) report- ed daily consumption of walnut lowers risk of heart diseases. Higher anti-oxidant and elevated levels of the fat fraction in walnut are important for heart health. Brain health. Brain function is immensely advanced by eating nuts. Walnuts are also associated with alleviation of depression and age-linked dis- eases (Grosso and Estruch, 2016). Arab and Ang (2015) reported daily consumption of walnuts enhance memory retention. Walnuts help reduce problems in metabolic syn- drome. Metabolic syndrome is complex disorder encompassing increased triglycerides, high blood Nutrients Total amount /100 gm walnut Calories 654 Water 4% Protein 15.2 g Carbs 13.7 g Sugar 2.6 g Sucrose 2.4 g Glucose 0.1 g Fructose 0.1 g Fiber 6.7 g Fats 65.2 g Saturated 6.13 g 16:00 4404 mg 18:00 1659 mg Monounsaturated fatty acids 8.933 g 18:01 8799 mg 20:01 134 mg Polyunsaturated fatty acids 47.174 g Omega-3 9.08 g Omega-6 38.09 g Trans fat 0 Vitamins Vitamin A 1 µg Vitamin C 1.3 mg Vitamin D 0 µg Vitamin E 0.7 mg Vitamin K 2.7 µg Vitamin B1 (Thiamine) 0.34 mg Vitamin B2 (Riboflavin) 0.15 mg Vitamin B3 (Niacin) 1.13 mg Vitamin B5 (Panthothenic acid) 0.57 mg Vitamin B6 (Pyridoxine) 0.54 mg Vitamin B12 0 µg Folate 98 µg Choline 39.2 mg Minerals Calcium 98 mg Iron 2.91 mg Magnesium 158 mg Phosphorus 346 mg Potassium 441 mg Sodium 2 mg Zinc 3.09 mg Copper 1.59 mg Manganese 3.41 mg Selenium 4.9 µg Cholesterol 0 mg Table 5 - Nutritional profile of Walnut Shah et al. - Bio-techniques for improvement of qualitative and quantitative traits in Walnut. A review 123 pressure, insufficient high-density lipoproteins, cho- lesterol, and obesity. Saneei et al. (2013) reported daily consumption of one ounce of walnuts for 2-3 months can lower risk of MetS- associated disorders. Benefits in treatment of type 2 diabetes. Type 2 diabetes is a growing problem in developed and industrialized countries. A better understanding of dietary changes is needed to improve this disorder. The significant diet regime for type 2 diabetes patients for lowering cardiovascular issues has been reported by consumption of walnuts on daily basis (Farr et al., 2017). Anti-cancerous effects. Walnuts contain a wide variety of antioxidant and anti-inflammatory bioac- tive components that may have anti-cancerous prop- erties (Byerley et al., 2017). Presence of polyphenolic compounds, phytosterols, gamma-tocopherol, omega-3 fatty acids and ellagic acid decline threat of chronic oxidative stress and ameliorate inflammatory properties to counteract intimidation of cancer advancement (Perugu and Vemula, 2014). Choi et al. (2016) and Al-Mahmood et al. (2016) reported a lower incidence of colon, breast and prostate cancer associated with walnut consumption. Other health benefits. Walnut nutrients exhibit significant function in the maintenance of bone struc- ture (Kajarabille et al., 2013). It has been recently confirmed that walnut consumption decline blood levels of N-terminal telopeptideNTx of type 1 colla- gen. More collagen composition signifies higher bone stability as well as lower mineral loss from bones (Griel et al., 2007). Katz et al. (2012) confirmed the potential significance of walnut consumption in body weight management. 9. Application of molecular markers in horticultural crops Horticultural crops are important for dietary pur- pose and source of revenue for farmers in the emerg- ing countries (Behera and France, 2016). Genetic Table 6 - Health effects of walnut Cardiovascular aspect Walnut benefit Blood quality Lowering LDL, the “bad” cholesterol; decreased total cholesterol; increased gamma-tocophe- rol; increased omega-3 fatty acids in red blood cells (alpha-linolenic acid) Vasomotor tone Decreased aortic endothelin; improved endothelial cell function Risk of excessive clotting Decreased maximum platelet aggregation rate; decreased platelet activation Risk of excessive inflammation Decreased C reactive protein (CRP); decreased tumor necrosis factor alpha (TNF-a) Blood vessels Improves the function of blood vessels by cutting the risk of plaque buildup in the arteries Brain tonic Relieves depression and age-related decline in brain function Decline in metabolic syndrome Reduction of various MetS-related problems Treatment of type 2 diabetes Regulates blood sugar and insulin metabolism Development of body structure Decrease blood levels of N-telopeptides of type 1 collagen (NTx); weight maintenance and prevention of obesity Anti-cancer activities Anti-inflammatory properties help lower risk of chronic inflammation Adv. Hort. Sci., 2018 32(1): 113-135 124 improvement in horticultural crops is not yet as note- worthy as it has been achieved in case of cereals and grasses. The advancement and accessibility of genomic resources in these crops can be utilized resourcefully to conserve and investigate existing genetic diversity and to understand the association between genotype-phenotype and to accelerate breeding (Chaturvedi and Sahijram, 2015). Current progress in mechanization and high-throughput sequencing can be utilized to decipher ambiguous and intricate genomes. Gene pyramiding and poly- genic resistance evaluation in diverse genotypes with extensive resistance have been made possible through the advent of molecular markers (Ansari, 2015). Walnut is valued for its wood and nut, phyto- chemicals but so far as its transcriptomics and genomic data is concerned, it is very limited (Hu et al., 2016). From the past few decades, a significant progress has been achieved by the use of molecular markers in plant biotechnology and unraveling their genomic intricacies for detecting and exploiting DNA polymor- phism in the plants. There are different markers, i.e. markers based on the morphology of plants, on bio- chemical functions and DNA-based molecular mark- ers (Parveen et al., 2016). These molecular markers are classified as hybridization-based or non-PCR- based markers. By using, these DNA based molecular markers, one can get complete information about a particular plant from the level of the nucleotide to a segment on DNA and frequencies of alleles, popula- tion structure and distribution of genetic diversity. Molecular markers explicitly reveal the polymor- phism at DNA level (Deoxyribonucleic acid). They are routinely used in various crop improvement pro- grams, ecological, physiological, genetic studies of plants and to progress the effectiveness and accuracy of traditional plant breeding through marker assisted selection (Allwright and Taylor, 2016). Important information for genetic diversity can be evaluated for different horticultural crops by using RAPD markers, for example, eggplant and watermelon (Trivedi et al., 2016), Moringa oleifera (L.) (Kumar et al., 2017). AFLP markers have effectively been utilized for evalu- ating genetic diversity in horticultural crops like Celosia argentea (Olawuyi et al., 2016), Rosa platya- cantha (Yang et al., 2016) and olive (Mnasri et al., 2017). Owing to their hyper variability and compe- tence in distinguishing of polymorphisms, SSR have become ideal markers for assessment of genetic diversity in horticultural crops l ike potato (Ghebreslassie et al., 2016), grapes (Rao, 2017), high density genetic map construction (Xue et al., 2008), population and conservation genetic studies, clonal identification (Liu et al., 2016 a, b), controlled crosses certification, species and hybrid identification, pater- nity determination (De La Rosa et al., 2013), marker assisted selection (Ashraf and Foolad, 2013) and genotyping (Billot et al., 2013). ISSR is extensively uti- lized in population genetics of horticultural crops i.e., Elaeis guineensis (Chagas et al. , 2015) and Rhododendron triflorum (Xu et al., 2017) and deter- mining the genetic diversity of horticultural crops such as bael (Aegle marmelos Corr.) (Mujeeb et al., 2017). This section of the chapter provides a detailed account of the exploitation of different molecular markers used in case of walnut study. Use of some like random amplified polymorphic DNA (RAPD), sim- ple sequence repeat (SSR), inter-simple sequence repeat (ISSR), single Nucleotide Polymorphism (SNP) are briefly discussed in improving, increasing the quality and quantity of walnut is summarized. Single Nucleotide Polymorphism (SNPs) have been widely used in plant germplasm management and breeding of fruit tree crops (van Nocker and Gardiner, 2014). These benefits have resulted in SNPs gradually becoming the markers of choice for exact genotype identification and diversity analysis in horticultural crops such as cacao (Theobroma cacao) (Fang et al., 2014 a), grapevine (Vitis vinifera) (Cabezas et al., 2011), pummelo (Citrus maxima) (Wu et al., 2014), strawberry (Fragaria spp.) (Longhi et al., 2014) and tea (Camellia sinensis) (Fang et al., 2014 b). 10. Molecular characterization in walnut Molecular characterization of walnut germplasm is mandatory requirement for establishing breeding purposes and establishment of proprietary rights. Conventional approaches for germplasm characteri- zation are evaluated based on comparative morpho- logical analysis. Due to the ecological effects on phe- notypic expression together with juvenile phase of walnut, there are ample hindrances in proper classifi- cation of walnut. To surmount these restrictions, molecular markers are employed for discrimination and identification of walnut accessions. These DNA based markers are not influenced by environment and can be identified in all development stages of plant tissues. Characterization of genetic variability in walnut was done using isozymes (Malvolti et al., 1994), to identify interspecific hybrids (Arulsekar et Shah et al. - Bio-techniques for improvement of qualitative and quantitative traits in Walnut. A review 125 al., 1985), in species and cultivars differentiation (Vyas et al., 2003) and to evaluate mating aspects (Rink et al., 1994). RFLP markers has been employed for evaluating parentage and ascertaining phyloge- netic associations and discriminate among species and cultivars in the genus Juglans (Aly et al., 1992; Fjellstrom et al., 1994; Fjellstrom and Parfitt, 1995). Description of various molecular markers used in molecular characterization of various walnut geno- types is given in Table 7. Randomly amplified polymorphic DNA (RAPD) RAPD is immensely significant in deciphering closely associated cultivars in walnut and results obtained are harmonized with preexisting data pro- cured from RFLPs owing to more vigorous polymor- phism exhibited by RAPD (Nicese et al., 1998). Furthermore, RAPD markers were also employed to assess polymorphism intensity at interspecific level among Persian walnut (J. regia) and in the Northern California black walnut [J. hindsii (Jeps.)] (Woeste et al., 1996 a) and to recognize a marker associated with hypersensitivity to the cherry leaf-roll virus (Woeste et al., 1996 b). Nicese et al. (1998) evaluated eighteen RAPD primers among nineteen walnut genotypes that comprised of strongly associated cul- tivars and parents in breeding programs. Following cluster analysis of closely associated genotypes, genotypes can be segregated into two classes having association with their closely related alleles. RAPD markers can investigate polymorphism to decipher variation among walnut genotypes particularly strongly associated genotypes and genetic similarity subsisting on RAPDs revealing can detect enough polymorphism to differentiate among walnut geno- types, even among closely identified (Hayward et al., 2015). RAPD markers are highly useful for evaluating genetic variability in genus Juglans through DNA fin- gerprinting to differentiate between valuable geno- types for selection (Pop et al., 2010; Ahmed et al., 2012). RAPD was employed to evaluate association among eight walnut genotypes thriving in Turkey and discrimination among indigenous and exotic geno- types together with their early bearing progenies (Erturk and Dalkilic, 2011). Xu et al. (2012 b) illustrat- Table 7 - Description of various molecular markers used in molecular characterisation of various walnut genotypes Walnut species/variety Molecular marker used Traits associated References Juglans regia RFLP Inheritance and linkage Fjellstrom et al., 1994 Juglans regia RAPD and SSR Phenotypic Pop et al., 2013 Juglans regia SSR Morphological Mahmoodi et al., 2013 Juglans regia SSR Morphological Karimi et al., 2014 Butternut (Juglans cinerea L.), Japanese Walnut (Juglans ailantifolia) and Buartnut (Juglans cinerea × Juglans ailantifolia) SSR Nut phenotype Chen et al., 2014 Juglans regia RAPD Butternut canker disease resistance Zhao et al., 2014 Juglans nigra, Juglans regia, and hybrid (Juglans x intermedia (Carr) RAPD Association between native/foreign genotypes with their early-bearing natural hybrids Erturk and Dalkilic, 2011 Juglans regia SSR Identification of interspecific hybrid Pollegioni et al., 2014 Juglans regia SSR Morphological Ebrahimi et al., 2011 Juglans regia ISSR Morphological and biochemical traits Malvolti et al., 2010 Juglans regia ISSR Distinction between native and internatio- nal cultivated genotypes Christopoulos et al., 2010 Juglans regia SLAF Disease resistance to anthracnose Zhu et al., 2015. Juglans regia AFLP Geographical proximity Bayazit et al., 2007 Juglans regia SNP Fingerprinting of 30 walnut genotypes Ciarmiello et al., 2013 Juglans regia SNP Genome sequencing Liao et al., 2014 Adv. Hort. Sci., 2018 32(1): 113-135 126 ed RAPD and AFLP for deciphering genetic diversity of walnuts in western Sichuan plateau and Qinba mountainous regions. The walnut genotypes were procured from 8 different regions and 32 RAPD primers and 28 AFLP primers in combination were recognized with polymorphic bands in entire walnut. In this study, high allelic number and elevated genet- ic diversity identified with RAPD and AFLP signify western China has significant genetic diversity resource and profuse genetic variance among wal- nuts. Zhao et al. (2014) employed RAPD as chief mol- ecular implement for characterizing genome of but- ternut (Juglans cinerea), Japanese walnut (Juglans ailantifolia), black walnut (Juglans nigra), Persian walnut (Juglans regia), Manchurian walnut (Juglans mandshurica), and an interspecific hybrid (J. ailan- tifolia cinerea) for species-specific markers. The study revealed 38 amplicons were exclusive to Japanese walnut and buartnut hybrids lacking in butternut. RAPD markers were relatively insufficient to differentiate intraspecific variability inside Japanese walnut, butternut, or their hybrids. RAPD can also decipher hybrid lineage as in the case for discriminating whether Japanese walnut or Manchurian walnut was the progenitor of hybrid line or not. The results demonstrated that some of the hybrids can be distinguished through morphological and genetic resemblances through RAPD as it demonstrates pedigree data. The resulting RAPD markers provide their utility in the conservation of butternut, an endangered North American species. Simple sequence repeat (SSR) SSR is enumerated among most precise molecular marker owing to their high polymorphism, co-domi- nant transmission, higher reproducibility, exhibiting higher resolution and more convenient PCR detec- tion in deciphering genetic relationship (Miah et al.. 2013). Wang et al. (2015), Ali et al. (2016), Ebrahimi et al. (2016), and Pang et al. (2017) illustrated research reports where in genetic characterization of walnut was evaluated using SSR. Han et al. (2016) described genetic diversity and population structure study of J. regia germplasm using ten primers devel- oped from expressed sequence tags (EST-SSR) and sequence polymorphisms among the phenylalanine ammonia lyase (PAL) gene. The result showed high level of population differentiation. In addition, SSR markers informative in Juglans regia may also be polymorphic in other Juglans (L.) species (Aldrich et al., 2003) and have utility for breeding hybrid root- stocks. SSR are more consistent and trustworthy sys- tem for molecular characterization. SSR has wide numerous relevance in case of walnut comprising of cultivar characterization, cultivar identification, pedi- gree substantiation for cultivar and in deciphering evaluation of superior rootstock for breeding and paternity analysis (Dangl et al., 2005). Ruiz-Garcia et al. (2011) employed 32 SSR primer pairs to character- ize 57 walnut cultivars originated from Spain and USA. In this research reports, 32 primer pairs flanking simple sequence repeats, were established in Juglans nigra to screen elite variety with high rate of poly- morphism. Further selection of 19 selected microsatellite markers provided differentiation of evaluated cultivars exhibiting 97 alleles and on an average 5 alleles per locus establishing SSR as prima- ry choice for characterization of walnut germplasm. The data generated from SSR characterization further confirmed molecular parameters of Spanish walnut to be different from Californian genotypes. Ahmed et al. (2012) illustrated genetic association of 82 walnut genotypes using 13 SSR and 20 RAPD primers exhibit higher level of genetic diversity in these walnut culti- vars thriving rigorously in climate of Jammu and Kashmir. These results demonstrate applications of SSR markers in walnut breeding and conservation. Pollegioni et al. (2014) illustrated genetic diversity and spatial genetic structure of 39 autochthonous Persian walnut populations analyzed transversely along Asian range via 14 neutral microsatellite mark- ers. Shah et al. (2016) reported genetic characteriza- tion of 96 walnut genotypes growing in North west- ern Himalaya as investigated by 19 SSR markers and the study displayed high polymorphism rate of 89.6%. Chen et al. (2014) demonstrated potential of SSR markers in the cultivar characterization together with intellectual property rights. Topcu et al. (2015) developed genomic libraries augmented with CA, GA, AAC, and AAG repeats via genomic DNA from J. regia cv. Maraş-18 to develop SSR markers for walnut. This study confirmed GA-enriched library as preferred alternative in terms of allelic number, polymorphism, productivity, and information content. Inter simple sequence repeat (ISSR) markers ISSR-PCR is a most convenient approach that sur- mounts most of these restrictions encountered by other molecular markers (Karimi et al., 2014). ISSR is most popular among plant improvement. Potter et al. (2002) has demonstrated the application of inter- simple sequence repeat (ISSR) markers in genetic characterization of English or Persian walnut (Juglans regia L.). In this study eight ISSR primers presented exclusive fingerprint for 48 cultivars investigated. The dendrogram developed from data presented classes Shah et al. - Bio-techniques for improvement of qualitative and quantitative traits in Walnut. A review 127 pertaining to known pedigrees which does not pro- vide this data anticipating the fact that there is restriction in context to utility of ISSR in determining genetic association among species. Christopoulos et al. (2010) reported immense application of ISSR markers in genetic characterization for deciphering genetic diversity among Greek natives of walnut (Juglans regia L.). In this study, similarity coefficient values signified presence of a higher extent of genet- ic variability. Majority of international cultivars were classified together whilst majority of Greek native populations cannot be classified in separate class. International cultivars exhibit lesser diversity than Greek native population genotypes. The pairwise regional PhiPT values signified that most geographi- cally isolated regions are the most genetically differ- entiated. The intense variability in Greek germplasm along with their desired traits anticipated that native germplasm can be utilized for breeding purposes and preservation of walnut germplasm. Li et al. (2011) employed ISSR markers to evaluate the genetic varia- tion and genetic structure to provide a theoretical basis and technical support for appropriate conserva- tion and application of existing genetic resources of walnut. Analysis of ANOVA (Analysis of variance) also showed genetic variance among populations was larger than within a population. Mantel test and UPGMA (Unweighted Pair Group Method with Arithmetic Mean) dendrogram based Nei’s genetic distance displayed that genetic distance between populations had more significant correlation with geographic distance. Ji et al. (2014) developed ISSR primers to assess the degree and design of genetic diversity among eight populations of North China Mountain Walnut (NCMW). This diversity was further confirmed through ANOVA analysis. Structure of NCMW and UPGMA cluster analysis demonstrated restricted gene flow, habitat devastation and geo- graphical isolation might be determining factors for population structure. UPGMA cluster further signify that eight populations of walnut can be classified into three discreet groups as per similarity coefficient and geographic origin but exhibited significant associa- tion with morphological traits especially nuts. Hu et al. (2016) in their classical experiments use transcrip- tomic information from RNA-Seq to understand development of polymorphic simple sequence repeats (SSRs, microsatellites) for understanding the population genetics of walnut. They studied more than 47.7 million clean reads, 99,869 unigenes having length of 747 bp. They further identified 39,708 (42.32%) genes, 63 new transcriptome-derived microsatellite markers. The identification and charac- terization of microsatellite markers in their study could help to explore the diversity, genetic structure, population genetics for improved walnut future breeding practices, besides providing a useful genetic resource information for studying and understanding the genomic and transcriptome aspect of walnut. Doğan et al. (2014) evaluated genetic association of 59 walnuts (Juglans regia L.) genotypes, international and Turkish by using three different types of molecu- lar markers i.e. RAPD, ISSR and SSR primers. These results exhibited that SSR markers offer elevated rank of polymorphism as compared to RAPD and ISSR. Single nucleotide polymorphism (SNP) markers SNP is the most abundant type of DNA variation in most species. The introduction of next generation sequencing (NGS), together with high throughput genotyping technology, makes it relatively easy to identify and use SNPs (Elshire et al., 2011). The new generation of molecular markers based on single nucleotide polymorphisms (SNP) represent a promis- ing and effective tool for fast and accurate species identification. Ciarmiello et al. (2013) developed a simple amplification refractory mutation technique, based on SNP markers of rDNA and cox2 intron I sequences, to fingerprint 30 walnut genotypes. rDNA sequences revealed the presence of 402 variations and Cox2 intron I sequences showed 769 variable positions. The findings revealed that the cox2 intron I region, either alone or in conjunction with rDNA, could be used effectively in identifying these walnut genotypes. Liao et al. (2014) performed genome sequencing of walnut and then all the sequence reads were mapped against genome assembly of wal- nut. In total, 49,202 nucleotide variations were detected including 48,165 single nucleotide polymor- phisms (SNP5) and 1037 insertions/deletions (InDels). 11. Conclusions The nutritional factors of walnut are improved due to the presence of numerous micro elements. The notable nutritive feature of walnuts relates to a rich range of polyphenols. The breeding character of walnut has bestowed it an inclusive diversity in genetic features. These results have been attained after an extended progression of evolution and by going through intricate environmental abnormalities. However, these techniques are susceptible to the environmental variations. The markers are adequate Adv. Hort. Sci., 2018 32(1): 113-135 128 in number and there is little or no diversity in the method employed for research on walnuts. Despite basic research method, the organization of informa- tion, its retrieval and presentation structures, form elaboration experienced immense advancement via molecular markers such as RFLP, ISSR, RAPD AFLP, SSR and SNP. This assessment offers data regarding health benefits of walnut at the global level and existing applications in the horticulture. The present work comprehensively describes the utilization of molecular markers in walnut plants which could help to improve/enhance the traits linked to its opti- mum/yield growth and sustainable production of innumerable essential metabolites having the ability to find applications in horticulture and allied science (Fig. 4). 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