Morphological to Molecular Markers: Plant Genetic Diversity Studies in Walnut (Juglans regia L.)—A Review

Persian walnut populations have tremendous morphological and allelic diversity in their germplasm due to heavy outcrossing and years of seed multiplication. These variations are assessed by morphological, cytological, biochemical, and molecular markers. Various researchers have used different tree, foliage, flower, nut, and kernel traits to evaluate morphological/phenotypic diversity. In walnut, morphological indices are first taken into consideration to describe and classify the germplasm, but the environment influences them. In comparison, DNA-based markers can detect genetic diversity at any stage of plant development and have been shown to be a potential tool for assessing variation at the DNA level and deciphering genetic relationships within and between species. Microsatellites are very powerful and informative among DNA-based markers in studying genetic relationships and genetic identity at different levels. They are neutral, highly frequent, uniformly distributed, hypervariable, codominant, highly reproducible, produce many alleles per locus, and require a small amount of DNA for analysis. Current breeding objectives can be achieved by selecting superior genotypes from the germplasm, supplemented by molecular characterization in the selection of parents for each breeding program. Therefore, the use of morphological and molecular markers is recommended for efficient exploration and utilization of germplasm resources and to improve diversity among genetic resources. The published literature on morphological and molecular markers, especially simple sequence repeats (SSRs), is presented in this review to provide current insights into the level of genetic diversity in walnut.

The walnut species (Juglans regia L.) belonging to the family of Juglandaceae (Bozhuyuk et al. 2020;Skender et al. 2020) are known to be among the oldest domesticated fruit species in the world.The name walnut is derived from the old English word "wealhhnutu", which means "foreign nut".Walnut trees have high adaptability to different climatic conditions, especially in temperate climates (Bernard et al. 2018b;Okatan et al. 2021).Walnut have a broad genetic diversity and a vast differentiation exists in walnut germplasm (Li-xue 2005).The vast genetic variation arises mainly from seed-based plantations and high heterozygosity (Büyüksolak et al. 2020;Wan et al. 2021).It is widely cultivated for its wood and nutrient-rich kernels (Bayazit et al. 2007), especially for its high antioxidant and omega-3 fatty acid content (Rahimipanah et al. 2010).As a result, walnuts are in high demand worldwide (Hassankhah et al. 2017;Shah et al. 2022).Globally, walnuts are second only to almonds in tree nut production and are among the priority crop groups listed by the FAO (Raja et al. 2017).The by-products of walnut, such as the hard shell and green husks, are used for the production of bioethanol, nut charcoal, jams, and alcoholic beverages (Jahanban-Esfahlan and Amarowicz 2018; Lancefield et al. 2017;Stampar et al. 2006;Wahid et al. 2017).The annual global walnut production in 2021 was 3500.18 k tons on an area of 1137.79 k ha (http://faostat3.fao.Org/home/.).At present, China contributes 43.31% to the total global production and is a key walnut-producing country.The USA, Iran, Turkey, Mexico, and India contributed 16.74%, 11.19%, 5.87%, 4.35%, and 0.88% to the total production, respectively.

Origin and Distribution
There is controversy over the origin of J. regia L. Fossil evidence, for example, points to America as the origin of walnuts.However, some previous reports point to Asia as the primary center of origin (Arzani et al. 2008).Other studies suggest that walnuts originated independently in Asia and Europe (Pollegioni et al. 2017(Pollegioni et al. , 2015)).Later, whole-genome sequencing data revealed the hybrid origin of Persian walnut between butternut and black walnut and its diversification in Asia (Zhang et al. 2019).Natural hybridization between wild and cultivated in Julandaceae was previously demonstrated by Wang et al. (2015b] and Zhao et al. (2018).The spread of walnuts to new habitats is believed to be due to trade and cultural dispersal (Feng et al. 2018).J. regia is distributed in China's Xinjiang province, Kyrgyzstan, Turkmenistan, Uzbekistan, the northwestern Himalayan regions of India and Pakistan, the mountains of Nepal and Tibet, and Afghanistan, Iran, Azerbaijan, Armenia, Georgia, and Turkey (Aradhya et al. 2009).The taxonomic studies based on fruit morphology and leaf architecture accept three or four sections with seven to 45 species of the genus Juglans L. (Juglandaceae) (Aradhya et al. 2007).Most species ( 16) are distributed in North, Central, and South America.In the Northern Hemisphere, it is found mostly in temperate and subtropical regions, with some species in Central America and along the Andes in South America (Bernard et al. 2018b;Pollegioni et al. 2015).Walnut have been widely cultivated in the Mediterranean region since the Pliocene, as evidenced by the fossil record.In contrast to fossil evidence, a recent study using non-coding intergenic spacer (NCS) portions of chloroplast DNA confirmed section Rhysocaryon as the youngest lineage within the genus Juglans and section Dioscaryon as the oldest (Aradhya et al. 2007).Recently, a walnut single nucleotide polymorphism (SNP) chip was developed (Marrano et al. 2019a) that can be used to answer previous questions about walnut evolution and origin, in addition to mapping economic traits.

Diversity Assessment and Analysis Techniques
The assessment of germplasm is important for future crop improvement programmes (Akça et al. 2020;Pop et al. 2013), and examining such resources for different characteristics generates useful information (Radivojevic et al. 2014) to choose beneficial genetic resources (Ebrahimi et al. 2015;Hassani et al. 2020).For instance, different investigators have employed various variables for the collection and conservation of promising genotypes such as spring frost resistance (Khadivi et al. 2019b) and nut traits (Khadivi-Khub et al. 2015a;Khadivi-Khub et al. 2015b).Morphological, cytological, and biochemical traits have been used to examine diversity, but such assessments are influenced by environmental conditions and the genetic makeup of a genotype (Vyas et al. 2003;Zeneli et al. 2005).Yet, they are essential in assessing diversity and choosing a genotype with superior economic qualities.However, for complex traits, it may prove inefficient.Therefore, the transition from traditional to molecular diversity analysis in the current genomics era has accelerated breeding and release of new varieties through accurate selection of superior genotypes.

Morphological Markers
Morphological markers are routinely used to assess diversity in walnuts.To assess this diversity, growth traits such as tree vigour, growth habit, density of branches, and colour of annual shoots are routinely used for evaluation by researchers (Khadivi-Khub 2014;Lotfi et al. 2019;Shah et al. 2021).Among the phenological traits, leaf bud emergence, timing of male and female flowering, and harvest date are used to assess walnut genetic diversity (Khadivi-Khub et al. 2015b;Khadivi et al. 2019a;Mahmoodi et al. 2016).However, other researchers have used traits such as inflorescences, fruit traits such as lateral bud flowering, type of dichogamy, frequency of male and female inflorescences, and alternate bearing (Norouzi et al. 2013;Poggetti et al. 2017;Rezaei et al. 2018).The outcrossing nature and long years of seed propagation in centers of origin have led to significant diversity in nut and kernel traits (Arab et al. 2019;Bujdoso and Cseke 2021;Khadivi et al. 2019a;Shah et al. 2021), phenology (Cosmulescu and Ionescu 2021;Hassankhah et al. 2017), lipid content (Çaǧlarırmak 2003;Shah et al. 2022), bud break (Arzani et al. 2008), spring frost tolerance (Mahmoodi et al. 2016), disease tolerance (Botu et al. 2001;Olson and Buchner 2002), dichogamy (Ebrahimi et al. 2015;Ghasemi et al. 2012), and lateralbearing (Botu et al. 2010;Rouskas and Zakynthinos 2001;Solar and Štampar 2003).Among these traits, nut and kernel traits are crucial for identifying superior-quality genotypes K (Arzani et al. 2008;Bhat and Ahmed 2003;Mahmoodi et al. 2016;Shah et al. 2021).For instance, the lateral-bearing walnut cultivar 'Chandler' was chosen for its excellent kernel quality and is currently the subject of research in a number of nations for the creation of superior cultivars (Bernard et al. 2018b).
Extensive germplasm studies have been conducted in regions such as Iran (Ebrahimi et al. 2015;Khadivi-Khub 2014;Rezaei et al. 2018), Turkey (Akca et al. 2015;Ipek et al. 2018), northwestern Himalayas of Jammu and Kashmir, India (Angmo et al. 2015;Shah et al. 2021;Shah et al. 2018;Sharma et al. 2014), and other countries as listed (Table S1).To comprehend the diversity of walnut, particularly in the northern Himalayan region where walnut is highly diverse, the DUS features developed for walnut (International Plant Genetic Resources 1994) must be evaluated (Rana et al. 2007a, b;Shah et al. 2021).Being a region closer to the origin of walnut, there is a need to study a large geographical area to identify superior walnut genotypes based on phenotypic traits.An attempt in this direction led to the identification of four genotypes which were later released as cultivars 'Hamdan' and 'Sulaiman' in Srinagar (Bhat and Ahmed 2003) and 'Parbat' and 'Bhushan' in Jammu.Similarly, promising French cultivars crossed with lateral bearing superior California cultivars led to the development of 20 new cultivars over the years (Marrano et al. 2019b).

Cytological and Biochemical Markers
In addition to morphological characteristics, cytological and biochemical markers, particularly isoenzymes and alloenzymes, have been used to infer walnut diversity.However, with the development of better molecular tools, their use in walnut genotyping has been limited.Cell karyotype and isoenzyme analysis (Fornari et al. 2001;Malvolti et al. 2001;Ouji et al. 2011;Vyas et al. 2003;Zeneli et al. 2005), allozymes (Busov et al. 2002;Vyas et al. 2003) are rapid methods for assessing diversity and require only a small amount of tissue for analysis.However, these methods are limited to analysing the diversity of walnut because of the limited numbers and low resolution of markers, and they are also sensitive to environmental factors (Bozhuyuk 2022).Cytological variation can be a powerful tool for assessing the genetic diversity of walnut as reported by several researchers in other plant species (Abedi et al. 2015;Esfahani et al. 2016).Among cytological parameters, nuclear DNA and an analytical tool such as flow cytometry are important tool for estimating the genome size of the genotype.Khorami et al. (2018) recently used genome size to predict nut weight and nut size of walnut trees.

Molecular Markers
Specific detectable DNA sequences connected with the trait of interest or linked genes on a given genome site are referred to as molecular markers.In comparison to conventional markers, DNA-based markers identify genetic variation within or among plant species (Cervera et al. 2000;Qu et al. 2011) and are unaffected by environment, type of tissue, or developmental stage (Di et al. 2006).Because of these benefits, the use of molecular markers in plant genetic diversity research has skyrocketed (Soriano et al. 2005).RFLP (Restriction Fragment Length Polymorphism), RAPDs (Random Amplified Polymorphic DNA) (Ahmed et al. 2012;Ku et al. 2011), ISSR (Inter Simple Sequence Repeats) (Christopoulos et al. 2010;Mahmoodi et al. 2012;Yang et al. 2007), AFLP (Amplified Fragment Length Polymorphism) (Andreakis et al. 2002;Bayazit et al. 2007;Kafkas et al. 2009), and SSRs (Simple Sequence Repeats) (Balapanov et al. 2019;Bernard et al. 2019;Bernard et al. 2020;Shah et al. 2020;Shah et al. 2018) have been used to distinguish walnut genotypes.Markers discriminate genotypes at the genetic level (Collard et al. 2005) due to differences in amplification band size, which may have resulted from nucleotide realignment or a mutation in the genome sequence.The recent construction of the reference genome 'Chandler' now allows researchers to identify distinct genotypes by examining differences in genome sequence alignments (Martínez-García et al. 2016;Stevens et al. 2018).Molecular markers provide a wealth of information about genome structure, organisation, and evolution (Cervera et al. 2000;Li et al. 2011).They are also useful for the mapping of qualitative and quantitative traits, linkage map construction (Crespel et al. 2002), germplasm improvement (Pearl et al. 2003), genotyping, parental identification, and population genetics (Pearl et al. 2003).The genome-wide association studies the identification and positioning of genes of important traits, which aid in MAS (Marker Assisted Selection) and gene cloning (De la Rosa et al. 2003;Ribeiro et al. 2011).The availability of SNP chips allows for the acceleration of walnut molecular breeding with the use of cutting-edge genomic techniques including genomic selection and association mapping (Marrano et al. 2019a).
Reference standard cultivars like 'Payne' (early season), 'Hartley' (midseason), and 'Franquette' (late season), as well as local check varieties, are frequently used to evaluate and analyze germplasm for phenological features due to their high variability under different climatic conditions.These traits can vary with harvest year, environmental conditions, horticultural practices, and genetic characteristics, altitude, planting direction, plant age (Simsek 2017;Simsek et al. 2017;Sütyemez et al. 2018).The majority of the Iranian genotypes have early to medium leafing (Khadivi-Khub 2014;Vahdati et al. 2015).Contrary to Turkish cultivars (Akca and Ozongun 2004), late-leafing is uncommon in Iran (Khadivi-Khub et al. 2015a).However, early bud break accessions are exposed to blight and spring frosts at higher altitudes (Aslantas 2006).In Iran, leaf opening occurred between 5 March and 2 May, while the blooming season extended from 29 March to 16 May (Alinia Ahandani et al. 2014;Khadivi-Khub et al. 2015b;Mahmoodi et al. 2019;Rezaei et al. 2018) and 2-9 April and second fortnight of April to 10 May, respectively, in Turkey (Cicek et al. 2020;Cosmulescu and Botu 2012;Cosmulescu and Ionescu 2021;Ipek et al. 2018;Simsek et al. 2010).Floral characteristics such as the nature of dichogamy and the abundance of male and female flowers, and the position of fruit buds on shoots are essential traits used in morphological characterization of walnuts (Sharma and Das 2003).Walnut plants are heterodichogamous in nature and genetic protandry is most common (Akca and Ozongun 2004;Bükücü et al. 2020;Ipek et al. 2018;Jaćimović et al. 2020;Khadivi-Khub 2014;Lotfi et al. 2019;Shah et al. 2021;Simsek 2017;Vahdati et al. 2015), but this may be affected by ecological conditions.However, Akhiani et al. (2017) reported homogamous nature and Tsampas and Botu (2013) protogynous.Because of this temporal variation in flowering, pollenizer is planted in commercial orchards to obtain successful pollination and fruit set (Soltész et al. 2003).

Molecular Diversity
Microsatellites are among the most common and widely dispersed DNA markers in the genome, are multi-allelic, can distinguish between homozygous and heterozygous loci, and are extremely repeatable (Foroni et al. 2005;Weising et al. 2005).These are extremely useful for studying genetic variability, genetic nature, and plant genetics (Simko 2008), and help preserve or establish quantitative variations within genomes (Frazer et al. 2004).They are crucial tools for connecting phylogenetic, physiological, and sequence-based physical mapping in plant species (Aggarwal et al. 2007;Kota et al. 2001).They are also very helpful in developing an optimal strategy for in-situ conservation (Gómez et al. 2004;Warburton and Hoisington 2001), for protected origin certification (Gemas et al. 2004), breeding programmes (Portis et al. 2004), or to better understand how indigenous plants adapt to different biological and environmental stresses (Farid et al. 2000).Because of their highly conserved flanking regions (Gupta and Varshney 2000), SSR markers are useful for screening of plant species at different levels of taxonomy (Hamza et al. 2004).In spite of the fact that they are very useful and aid in unrevealing the population structure (Dangl et al. 2005;Foroni et al. 2005Foroni et al. , 2007;;Victory et al. 2006;Woeste et al. 2002), very few SSRs have been developed so far.SSRs are developed and constructed through different means.SSRs created de novo are often species-specific, expensive, and time-intensive (Pashley 2006).However, EST (Expressed Sequence Tag)-based are more advantageous than de novo SSRs.SSRs were also constructed using genomic libraries enriched with di-and tri-nucleotide repeats from the genomic DNA of walnut cv.'Maras-18' (Topçu et al. 2015), BAC(Bacterial Artificial Chromosome)/YAC (Yeast Artificial Chromosome) libraries, and cDNA (Scott 2001).Recently, SSR markers have been discovered and created using the time-and money-saving FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) approach (Zane et al. 2002).The first research on walnuts using a group of SSR markers was published by Chen et al. (2014), Dangl et al. (2005), Foroni et al. (2005, 2007), Hoban et al. (2008), Najafi et al. (2014), Robichaud et al. (2006), Ross-Davis et al. (2008), Topçu et al. (2015), Victory et al. (2006), Woeste et al. (2002), and Zhang et al. (2010, 2013).About 1300 SSR markers have been screened so for in walnut, and most of them have been produced from J. regia.There are 56 SSRs from J. nigra, 20 from J. mandshurica (Chen et al. 2013), 892 from J. regia (Zhang et al. 2010(Zhang et al. , 2013)), 319 from the 'Chandler' BAC end sequences (Chen et al. 2014;Ikhsan et al. 2016), and 13 from J. cinerea (Eser et al. 2019).

SSR Molecular Genetic Diversity
Many molecular genetic marker metrics, such as the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), and expected heterozygosity (He), as well as the polymorphic information content (PIC), are used to quantify molecular genetic diversity (Mohsenipoor et al. 2009).By taking into account the number of alleles per locus and their relative frequencies in the population, PIC values can be used to determine the discriminatory potential of any locus.The PIC > 0.5 are considered informative markers and > 0.7 are good for mapping and both can contribute substantial knowledge to walnut breeding and genetics (Orhan et al. 2020).The numerous studies in walnut that used SSR molecular markers to examine genetic diversity are summarized in Table S2, along with their mean genetic diversity characteristics.The simplest technique to quantify genetic variation within genotypes/samples and populations is to count the number of alleles amplified.Victory et al. (2006) reported the highest average number of alleles per locus (23.8), whereas Zhang et al. (2010) found the lowest number (3).Other researchers found it to be 4.25-12 (Itoo et al., 2023;Balapanov et al. 2019;Bernard et al. 2018a;Dangl et al. 2005;Ebrahimi et al. 2011;Foroni et al. 2005Foroni et al. , 2007;;Karimi et al. 2010;Khokhlov et al. 2018;Kim et al. 2012;Mahmoodi et al. 2013;Orhan et al. 2020;Pollegioni et al. 2009Pollegioni et al. , 2011;;Pop et al. 2013;Vahdati et al. 2015;Wang et al. 2008).However, the low or higher number of alleles can be linked to the study group's narrow and wide nature of germplasm, intensity and nature of markers utilized, demography of the sampling area and other factors (Feng et al. 2018;Shah et al. 2020).For example, Feng et al. (2018) detected lower average alleles per locus (6.79) at EST-SSR loci than 12 and 14.2 alleles per locus reported by Aradhya et al. (2017) and Pollegioni et al. (2020) based on nuclear SSRs.In China, Feng et al. (2018) found lower heterozygosity than reported for J. regia (Bai et al. 2014;Han et al. 2016) and that of other Juglans species (Bai et al. 2014;Hu et al. 2017;Laricchia et al. 2015).In Iran, Vahdati et al. (2015) found high a number of alleles in comparison to levels of variability seen in European populations and China's central and western regions (Dangl et al. 2005;Wang et al. 2008).In recent investigations, observed homozygosity was 0.57-1.00(mean 0.80; Eser et al. ( 2019)), 0.633-0.895(mean (0.75); Balapanov et al. 2019), 0.548-0.927 (mean (0.803);Zhou et al. (2021)), 0.39-0.80(mean (0.60); Orhan et al. (2020)), 0.250-0.833(mean (0.514); Bujdoso and Cseke (2021)).However, Aradhya et al. (2017) reported around 0.5 across Eurasia and Pollegioni et al. (2017) found 0.559 among walnut populations from Central Asia, Western Asia and the Middle East.The expected hetrozygosity was found higher than the observed heterozygosity (Balapanov et al. 2019;Bernard et al. 2018a;Guney et al. 2021;Karimi et al. 2010;Khokhlov et al. 2018;Mahmoodi et al. 2013;Orhan et al. 2020;Pollegioni et al. 2011;Pop et al. 2013;Ruiz-Garcia et al. 2011;Vahdati et al. 2015), indicating a lack of heterozygotes in the genotypes or populations.This may be due to free inbreeding, selective mating, small sampling population size, and other factors.The utility of a PIC marker for detecting polymorphisims within populations and genotypes is determined by the number of detectable alleles and their frequency distribution (Guney et al. 2021).The PIC value in recent research studies ranged from 0.15 to 0.86.Guney et al. (2021) found it to be between 0.42 and 0.86 with an average of 0.68, whereas Orhan et al. (2020) found 0.54-0.85with an average of 0.68, Bernard et al. (2018a) found 0.15-0.75 with an average of 0.52, and Vahdati et al. (2015) 0.56-0.82with an average of 0.72.The variations in PIC value by different researchers may be due to use of different type and number of SSR markers, number and location of samples (Orhan et al. 2020).Following that, a number of researchers worked tirelessly to build polymorphic SSR markers.The genomic information gathered from the screening of multiple SSR markers in walnut genetic diversity research is presented in Table S2.
Pedigree analysis, breeding, population genetics, germplasm management, pollen flow analysis, and cultivar identification are just a few of the potential applications of microsatellites in walnut genetics (Farid et al. 2000;Gemas et al. 2004;Gómez et al. 2004).For example, the power of informativeness of microsatellite loci helped to distinguish prominent European cultivars from American cultivars (Foroni et al. 2006), Italian cultivars and genotypes (Foroni et al. 2005(Foroni et al. , 2007)), and other Juglans species (Aldrich et al. 2003).The J. regia L. cultivar, or other species used for the development of paradox hybrids, is ascertained by using SSRs (Potter et al. 2002).The production of a high number of private alleles helps for the identification of a genotype and all the cultivars by SSRs (Potter et al. 2002).SSR-based pedigree analysis assists researchers to better understand pollen transport inside and among walnut orchards, reducing production loss caused by blackline disease (Bernard et al. 2018b;Vahdati et al. 2021).The SSR's loci identified in J. nigra by (Woeste et al. 2002) were helpful in distinguishing the closely related cultivars except budsports (Dangl et al. 2005).Moreover, the SSR's developed from J. nigra L. yielded fewer alleles in Iranian populations (Karimi et al. 2010) as compared to J. nigra populations (Victory et al. 2006).SSRs have been found to exhibit a drop in allele numbers when used in similar species (Roa et al. 2000).However, in Tibet Wang et al. (2015a] found that markers used by K Dangl et al. (2005) and Woeste et al. (2002) produce more alleles in J. sigillata than J. regia and the levels of allele variability were more than that of reported by Dangl et al. (2005) and Pollegioni et al. (2011).The selection of favoured genotypes of Juglans regia L. having superior nut and kernel traits results in genetic drift and loss of alleles and fixation of others (Pollegioni et al. 2009;Wang et al. 2008).Notwithstanding, the genetic diversity of wild populations of J. regia L. in China was substantially lower than landrace populations (Wang et al. 2008).Previous research using chloroplast intergenic spacers (Aradhya et al. 2009) and SSR markers (Gunn et al. 2010) could not differentiate between J. regia and J. sigilata.Howerver, a recent study found that the two species can be well discriminated with the SSR loci (Wang et al. 2015a).The disparity in results may be because the previous studies did not look at the possibility of introgression between two species.SSR markers are useful for investigations in population genetics and conservation genetics, as well as the examination of variation between species and populations (Karimi et al. 2010;Woeste et al. 2002).SSRs revealed a high level of inter-population differentiation in Iranian J. regia while maintaining a distinct intra-population, which is attributed to its outcrossing nature (Vahdati et al. 2015).

Conclusions
In any breeding programme, genetic diversity is crucial for selecting parents for the continuous development of novel cultivars to fulfil the expanding needs of the growing population and international standards.As a result, the primary goal of any diversity assessment is to measure variation, identify and select superior plants, conservation of these seedling trees for future use.Although many diversity studies have been published from naturally occurring biodiversity hot spots, but relatively few studies have been conducted in non-accessible primary centres of diversity, such as the steep areas of northwestern Himalayas.These uncharted territories contain germplasm with a wide range of variants.Exploration in these areas will aid the breeder or researcher in identifying promising choices that can be used to develop local hybrids with better climatic adaptability, productivity, and quality.Although molecular markers, particularly SSRs, can play a key role in walnut improvement, genomic research, fingerprinting, genetic mapping, map-based cloning of candidate genes, and marker aided breeding, little work has been done to produce a large number of polymorphic markers.As a result, a larger number of SSR markers will be generated, allowing us to achieve the necessary crop improvement results by constructing linkage groups for commercially significant nut attributes.More wide sampling and thorough sampling using microsatellite markers will be used in future J. regia studies to assist in defining the population genetic architecture of Persian walnut in greater detail.