Landscape genetics of Persian walnut (Juglans regia L.) across its Asian range

Persian walnut (Juglans regia L) is an economically important species cultivated worldwide for its wood and nuts. Despite the increasing interest in the development of conservation strategies for walnut germplasm, an accurate and full-scale overview of wild genetic resources of J. regia has not been conducted because natural populations are located in regions of Asia historically difficult to access. In this study, we estimated the genetic diversity and spatial genetic structure of 39 autochthonous Persian walnut populations sampled across its Asian range using 14 neutral microsatellite markers. A landscape genetic overlay approach was applied to detect the areas of current reservoirs of walnut genetic diversity in the Asian range and to evaluate the role of landscape in shaping walnut genetic diversity since the Last Glacial Maximum. Although Persian walnut has been highly manipulated by humans over the last 2,000 years, we determined that patches of high genetic diversity still exist in the Caucasus and mountains of Central Asia where J. regia might have survived after Pleistocene glaciations. We detected a clear separation of Persian walnut into four main genetic clusters centered in (1) western Kyrgyzstan, (2) western and south–central Asia, (3) east–central Uzbekistan, and (4) Xinjiang and Shandong provinces (China). Overlay of maps showed a coincidence between groups of walnut populations and potential barriers to gene flow such as the Hindu Kush, Pamir, Tien Shan, and Himalaya mountains and the Karakum, Kyzyl Kum, and Taklamakan deserts. This study claimed the relevance of the preservation of walnut genetic resources in the Asian range.     

Walnut: past and future of genetic improvement

Persian or English walnut (Juglans regia L.), the walnut species cultivated for nut production, is one of the oldest food sources known. Persian walnuts, native to the mountain valleys of Central Asia, are grown worldwide in temperate areas. World production exceeds three million tons since 2012, mostly provided by China, the USA, and Iran. Despite very ancient culture of walnut species (Juglans spp.), breeding actually started in the twentieth century. Using a range of methodologies, from morphological markers to the most recent advances in genome analysis, many genetic studies of walnut have been conducted during the past 30 years, including examination of diversity, determination of relationships within or among germplasm collections and populations, phylogenetic and origin elucidation, genetic map construction, and biotic or abiotic stress investigations. The genetic improvement of walnut has undergone great evolution. The producing countries of the Middle East have widely studied morphological characteristics of walnut. The USA and France, for example, are behind important cultivar releases such as “Chandler” and “Franquette.” Finally, genomics represents a major breakthrough in walnut improvement, in particular by recent sequencing of both chloroplast and nuclear genomes. This review summarizes worldwide molecular and “omics” studies and gives an overview of the main walnut breeding programs.     

Ancient Humans Influenced the Current Spatial Genetic Structure of Common Walnut Populations in Asia

Common walnut (Juglans regia L) is an economically important species cultivated worldwide for its wood and nuts. It is generally accepted that J. regia survived and grew spontaneously in almost completely isolated stands in its Asian native range after the Last Glacial Maximum. Despite its natural geographic isolation, J. regia evolved over many centuries under the influence of human management and exploitation. We evaluated the hypothesis that the current distribution of natural genetic resources of common walnut in Asia is, at least in part, the product of ancient anthropogenic dispersal, human cultural interactions, and afforestation. Genetic analysis combined with ethno-linguistic and historical data indicated that ancient trade routes such as the Persian Royal Road and Silk Road enabled long-distance dispersal of J. regia from Iran and Trans-Caucasus to Central Asia, and from Western to Eastern China. Ancient commerce also disrupted the local spatial genetic structure of autochthonous walnut populations between Tashkent and Samarkand (Central-Eastern Uzbekistan), where the northern and central routes of the Northern Silk Road converged. A significant association between ancient language phyla and the genetic structure of walnut populations is reported even after adjustment for geographic distances that could have affected both walnut gene flow and human commerce over the centuries. Beyond the economic importance of common walnut, our study delineates an alternative approach for understanding how the genetic resources of long-lived perennial tree species may be affected by the interaction of geography and human history.     

Edible nuts of the pacific northwest

Only four kinds of native nut—hazelnut, chinquapin and two acorns—and introduced American walnut, Persian walnut and European filbert constitute the sources of edible nuts in this region.     

Using GFP as a Scorable Marker in Walnut Somatic Embryo Transformation

Somatic embryogenesis is the foundation of genetic transformationin several economically important tree species. In the Juglansregia L. (Persian walnut) somatic embryogenesis-based transformationsystem, a major limiting factor is the selection of non-chimerictransgenic embryos in tissue culture. We transformed Persianwalnut somatic embryos with the S65T synthetic green fluorescentprotein (GFP) gene in order to assess the effect of this visualmarker gene on embryo viability and the selection of transgenicembryos. Following a 10 d period of transient GFP expressionin all inoculated embryos, stable fluorescent sectors were apparentin several embryos, allowing efficient and rapid visual selectionof primary transgenic embryos. Two chimeric embryos were selected40 d after transformation, and these two embryos gave rise to13 stable transgenic embryo lines and 44 whole plants. GFP-expressingwalnut plants and embryos developed normally and transformationwas verified by GUS analysis. Our analysis suggests that theuse of GFP as a selectable marker can significantly reduce labour,cost, and time in the walnut somatic embryogenesis-based transformationsystem. Copyright 2000 Annals of Botany Company Juglans regia, Persian walnut, somatic embryogenesis, transformation, GFP, scorable marker, in-culture selection efficiency, ß-glucuronidase, fluorescence microscopy     

Phytopatological problems and solutions in the walnut orchards along Lake Balaton

European or Persian Walnut (Juglans regia) is an important and healthy food as well as base material of timber industry. Several pests (pathogens and insect pests) may cause serious damages on walnut. These are less known on the crop land of the tree. Results of some years of our experiments including bacteriological and mycological studies, are presented in this paper. The optimum time of chemical protection against the walnut blight (Xanthomonas arboricola pv. juglandis) was determined. Occurrences of pathogenic fungi were surveyed in an orchard and on home garden trees in Hungary (18 fungus species were identified). The following experimental results are reported on the pathogenic fungi: cultivar resistance to walnut anthracnose (Gnomonia leptostyla), dying of wood parts in the cultivar collection, application of the spore trap, in vitro fungicide testing against Phomopsis juglandina.     

Effect of exogenous ABA on somatic embryo maturation and germination in Persian walnut (<Emphasis Type="Italic">Juglans regia</Emphasis> L.)

Low efficiency of embryo maturation, germination and conversion to plantlets is a major problem in many species including Persian walnut. We studied the effects of abscisic acid (ABA) and sucrose, on the maturation and germination of Persian walnut (Juglans regia) somatic embryos. Individual globular somatic embryos were grown on a maturation medium supplemented with different combinations of ABA and sucrose for ca. 1 month, until shoot meristems and radicles had developed. White and opaque embryos in late cotyledonary stage were subjected to desiccation after the culture period on maturation media. The number of germinated somatic embryos was influenced by the concentrations of ABA in the maturation medium. The best treatment for germination, in which both shoot and root were developed contained 2 mg l⁻¹ ABA and resulted in 41% conversion of embryos into plantlets. Regeneration was reduced at higher levels of ABA. While ABA always reduced the rate of secondary embryogenesis, treatments containing 4.0% sucrose significantly increased the number of secondary embryos. On the other hand, sucrose had little influence on maturation. Normal and abnormal embryos were verified anatomically.     

Characterizing the walnut genome through analyses of BAC end sequences

Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots of J. regia cv. Chandler using the HindIII and MboI cloning sites. A total of 48,218 high-quality BAC end sequences (BESs) were generated, with an accumulated sequence length of 31.2?Mb, representing approximately 5.1% of the walnut genome. Analysis of repeat DNA content in BESs revealed that approximately 15.42% of the genome consists of known repetitive DNA, while walnut-unique repetitive DNA identified in this study constitutes 13.5% of the genome. Among the walnut-unique repetitive DNA, Julia SINE and JrTRIM elements represent the first identified walnut short interspersed element (SINE) and terminal-repeat retrotransposon in miniature (TRIM) element, respectively; both types of elements are abundant in the genome. As in other species, these SINEs and TRIM elements could be exploited for developing repeat DNA-based molecular markers in walnut. Simple sequence repeats (SSR) from BESs were analyzed and found to be more abundant in BESs than in expressed sequence tags. The density of SSR in the walnut genome analyzed was also slightly higher than that in poplar and papaya. Sequence analysis of BESs indicated that approximately 11.5% of the walnut genome represents a coding sequence. This study is an initial characterization of the walnut genome and provides the largest genomic resource currently available; as such, it will be a valuable tool in studies aimed at genetically improving walnut.     

Genetic Diversity of Xanthomonas arboricola pv. juglandis (synonyms: X. campestris pv. juglandis;X. juglandis pv. juglandis) Strains from Different Geographical Areas shown by Repetitive Polymerase Chain Reaction Genomic Fingerprinting

A world-wide collection of 61 Xanthomonas arboricola pv. juglandis strains, isolated from Persian walnut ( Juglans regia L.) or obtained from international culture collections and bacterial plant diseases laboratories, were studied by means of repetitive polymerase chain reaction (PCR) genomic fingerprinting using ERIC, BOX and REP primer sets and polyacrylamide gel electrophoresis. Cluster analyses were performed by UPGMA . Copper resistance, ability to hydrolize starch and quinate metabolism of the strains was also assessed. Pathogenicity was tested by inoculating leaves and nuts of Persian walnut seedlings. Polyacrylamide gel electrophoresis allowed very clear and reproducible differentiation of the PCR products. Cluster analysis showed the existence of three major groups of strains. The first two groups were 85% genetically similar, whereas the third clustered at 78% similarity with the other two. Each group could be divided into two subgroups which clustered according to the geographical origin of the isolates. In some cases, different genomic profiles were shown by strains from one country. This is possibly due to Persian walnut cultivation being mainly based on ecotypes and/or local seedlings that have become adapted to particular environments and so have allowed selection of different X.a . pv. juglandis populations. All strains were pathogenic and positive in starch hydrolysis and quinate metabolism tests. This is the first record of copper-resistant strains occurring outside California, USA.     

Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation

Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants.     

A new genomic tool for walnut (Juglans regia L.): development and validation of the high‐density Axiom™ J. regia 700K SNP genotyping array

Over the last 20 years, global production of Persian walnut (Juglans regia L.) has grown enormously, likely reflecting increased consumption due to its numerous benefits to human health. However, advances in genome‐wide association (GWA) studies and genomic selection (GS) for agronomically important traits in walnut remain limited due to the lack of powerful genomic tools. Here, we present the development and validation of a high‐density 700K single nucleotide polymorphism (SNP) array in Persian walnut. Over 609K high‐quality SNPs have been thoroughly selected from a set of 9.6 m genome‐wide variants, previously identified from the high‐depth re‐sequencing of 27 founders of the Walnut Improvement Program (WIP) of University of California, Davis. To validate the effectiveness of the array, we genotyped a collection of 1284 walnut trees, including 1167 progeny of 48 WIP families and 26 walnut cultivars. More than half of the SNPs (55.7%) fell in the highest quality class of ‘Poly High Resolution’ (PHR) polymorphisms, which were used to assess the WIP pedigree integrity. We identified 151 new parent‐offspring relationships, all confirmed with the Mendelian inheritance test. In addition, we explored the genetic variability among cultivars of different origin, revealing how the varieties from Europe and California were differentiated from Asian accessions. Both the reconstruction of the WIP pedigree and population structure analysis confirmed the effectiveness of the Applied Biosystems? Axiom? J. regia 700K SNP array, which initiates a novel genomic and advanced phase in walnut genetics and breeding.     

The identification and characterization of a genetic marker linked to hypersensitivity to the cherry leafroll virus in walnut

Walnut blackline disease, caused by the walnut strain of the cherry leafroll virus, causes fatal necrosis of the graft union between susceptible, infected scions of Persian walnut (Juglans regia L.) and hypersensitive, resistant rootstocks. A backcross breeding program to transfer hypersensitivity to cherry leafroll virus from the Norther California black walnut (Juglans hindsii (Jeps.)), into Persian walnut was begun in 1987. Hypersensitivity to the virus is inherited as a single, dominant gene. The current procedures for identifying hypersensitive backcross progeny are slow and labor intensive. Bulks of DNA from backcrosses that were either hypersensitive or susceptible to cherry leafroll virus were compared using randomly amplified polymorphic DNA. One random decamer, sequence 5-CTCCTGCCAA-3 (OP-K15), produces a polymorphic fragment of about 720 bp that has about 7% recombination with hypersensitivity to cherry leafroll virus in our backcross populations. The polymorphic fragment was cloned and converted into a restriction fragment length polymorphism to demonstrate that it is a distinct, low-copy sequence.     

Minor oil-producing crops of the United States

The pits and nuts of almond, Persian walnut, pecan, filbert, tung, apricot, prune, peach, cherry and plum; the fruit pulp of avocado and olive; and the seeds of citrus fruits, grape, apple, pear, cranberry and numerous other domestically cultivated plants are sources of valuable oils already in use.     

Genetic diversity and genetic structure of Persian walnut (<Emphasis Type="Italic">Juglans regia)</Emphasis> accessions from 14 European,African, and Asian countries using SSR markers

Persian walnut (Juglans regia L.) is the world’s most widely grown nut crop, but large-scale assessments and comparisons of the genetic diversity of the crop are notably lacking. To guide the conservation and utilization of Persian walnut genetic resources, genotypes (n = 189) from 25 different regions in 14 countries on three continents were sampled to investigate their genetic relationships and diversity using ten microsatellite (SSR) loci. The SSRs amplified from 3 to 25 alleles per locus, with a mean value of 11.5 alleles per locus. The mean values of observed and expected heterozygosity were 0.62 and 0.73, respectively. Based on Nei’s genetic identity, accessions from Bratislava (Slovakia) and Antalya (Turkey) showed the lowest similarity (0.36), while accessions from Algeria and Tunisia as well as accessions from Debrecen (Hungary) and Trnava (Slovakia) had the highest similarity (0.97). Two populations from Iran (Alborz and Ardabil) had the highest number of private alleles (7 and 5), but they were quite different as they also had the lowest genetic identity when compared to the remaining populations as well as to each other. Although overall differentiation among regions was relatively low (F _st  = 0.07), cluster analysis grouped accessions generally but not completely according to geography. STRUCTURE software confirmed these results and divided the accessions into two main groups, separating accessions collected from Europe and North Africa from those from Greece and the Near East. Results indicate the presence of a likely center of diversity for Persian walnut in Eastern and Southeastern Europe. They also provide information that can be used to devise conservation actions. Notably, the genetic diversity of threatened populations from two regions in Iran should be conserved.     

The walnut (Juglans regia) genome sequence reveals diversity in genes coding for the biosynthesis of non‐structural polyphenols

The Persian walnut (Juglans regia L.), a diploid species native to the mountainous regions of Central Asia, is the major walnut species cultivated for nut production and is one of the most widespread tree nut species in the world. The high nutritional value of J. regia nuts is associated with a rich array of polyphenolic compounds, whose complete biosynthetic pathways are still unknown. A J. regia genome sequence was obtained from the cultivar ‘Chandler’ to discover target genes and additional unknown genes. The 667‐Mbp genome was assembled using two different methods (SOAPdenovo2 and MaSuRCA), with an N50 scaffold size of 464 955 bp (based on a genome size of 606 Mbp), 221 640 contigs and a GC content of 37%. Annotation with MAKER‐P and other genomic resources yielded 32 498 gene models. Previous studies in walnut relying on tissue‐specific methods have only identified a single polyphenol oxidase (PPO) gene (JrPPO1). Enabled by the J. regia genome sequence, a second homolog of PPO (JrPPO2) was discovered. In addition, about 130 genes in the large gallate 1‐β‐glucosyltransferase (GGT) superfamily were detected. Specifically, two genes, JrGGT1 and JrGGT2, were significantly homologous to the GGT from Quercus robur (QrGGT), which is involved in the synthesis of 1‐O‐galloyl‐β‐d ‐glucose, a precursor for the synthesis of hydrolysable tannins. The reference genome for J. regia provides meaningful insight into the complex pathways required for the synthesis of polyphenols. The walnut genome sequence provides important tools and methods to accelerate breeding and to facilitate the genetic dissection of complex traits.     

The relationship between phenology of pistillate flower organogenesis and mode of heterodichogamy in Juglans regia L. (Juglandaceae)

 We investigated the degree of organogenesis completed at the end of the growing season in pistillate flowers of heterodichogamous Juglans regia, English or Persian walnut. Terminal buds from paired cultivars, one each protandrous and protogynous, chosen to represent early, midseason and late leafing walnuts, were examined by scanning electron microscopy. Results indicate that pistillate floral primordia in protandrous individuals had not progressed beyond involucre initiation during the season prior to bloom. In protogynous individuals, floral differentiation had progressed to the initiation of perianth primordia. These observations are compared with an earlier report on staminate flower differentiation in the same cultivars where a comparable, but opposite, relationship exists. We conclude that the degree of differentiation in both staminate and pistillate flowers that must be completed between the time growth resumes in the spring and anthesis is a developmental determinant of the mode of heterodichogamy in walnut. Received: 15 June 1996 / Revision accepted: 25 October 1996     

华北山区核桃液流变化特征及对不同时间尺度参考作物蒸散量的响应

核桃是华北山区重要的经济树种,但该地区水资源紧缺,因此研究核桃液流耗水变化及其对气象因子的响应对于加强核桃水分管理、确定水分承载力具有指导意义。2008—2010年连续3个生长季节观测分析了华北山区核桃单株液流变化特征,研究了不同时间尺度(日尺度、月尺度)核桃液流与气象因子、参考作物蒸散量(ET0)的关系,并利用2009—2010年数据建立了拟合方程,在此基础上用拟合方程的估算值与2008年液流实测值进行了对比验证。结果表明:核桃液流具有明显的时间变化特征,最大液流量出现在5、6月份,2008—2010年生长季节(4—9月份)核桃液流量分别为893.23 L、854.88 L和841.77 L,日平均液流量分别为4.96 L、4.75 L和4.68 L。液流年际差异主要是由年降雨量引起。核桃液流与不同时间尺度下气象因子、ET0均有较好的相关性,且随着时间尺度的增加,二者间的相关性变大。利用2009—2010年液流实测值与气象因子、ET0建立的拟合方程,对2008年液流值进行了拟合,并与实测值进行了对比验证,发现不同时间尺度下液流拟合值与实测值均具有较好的一致性。在不能实现对液流进行连续观测的情况下,可以结合当地的气象资料、ET0等因子,利用建立的拟合方程对树木液流进行估算,弥补缺失的数据,具有较高的精确性,可用于指导当地核桃水分管理。     

De novo assembly and characterization of transcriptome using Illumina sequencing and development of twenty five microsatellite markers for an endemic tree Juglans hopeiensis Hu in China

The Chinese walnut (Juglans hopeiensis Hu) is an endemic temperate tree species and narrowly distributed in China. However, there are still few specific molecular markers for understanding genetic diversity of this walnut. In this study, more than 44 million sequencing reads were generated using Illumina sequencing technology. De novo assembly yielded 93,822 unigenes with an average length of 731 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.3%) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 15,903 (17.0%) unigenes. To contribute to its conservation and management, twenty five microsatellite markers were identified in J. hopeiensis were screened for polymorphism across 27 Chinese walnut tree individuals from two locations. The number of alleles ranged from two to nine, observed heterozygosity ranged from 0.016 to 0.933 (mean 0.468), and expected heterozygosity from 0.022 to 0.823 (mean 0.462). The polymorphic information content (PIC) ranged from 0.012 to 0.831 (mean 0.437). The development of these new microsatellite markers will be useful for studying population genetic structure, evolutionary ecology, conservation, and genetic breeding of this endemic walnut tree or other Juglans species.     

Genetic diversity of superior Persian walnut genotypes in Azadshahr,Iran

Persian walnut (Juglans regia L.) is known to have originated in central and eastern Asia. Remnants of these wild populations can still be found in the Hyrcanian forest in north-eastern Iran. In this study, 102 individual walnut trees from four geographic populations in the Azadshahr province (Vamenan, Kashidar, Rudbar and Saidabad) were sampled. We characterized individual trees using 28 standard morphological traits. The range of traits varied widely for some economically important characteristics including nut weight (6.1–19.79 g), kernel weight (2.9–9.4 g), and kernel fill percentage (26.51–60.34%). After morphological evaluation, 39 superior individuals based on nut quality and kernel fill percentage were selected for further genetic analysis. Individual superior trees were genotyped using 10 simple sequence repeat markers (SSR) and genetic diversity. Number of alleles per locus ranged from 3 (WGA005) to 12 (WGA054). Clustering analysis of 10 SSR loci divided the genotypes into three main groups. PCoA analysis clearly sorted genotypes into one of four distinctive groups which aligned with the cluster analysis. All analyses showed that individuals from Saidabad were genetically distinct. Likewise, results indicated that the high level of genetic diversity in Azadshahr region walnuts may provide a diverse source for superior walnuts in walnut breeding programs.     

Response of the wheat rhizosphere soil nematode community in wheat/walnut intercropping system in Xinjiang,Northwest China

Intercropping Persian walnut (Juglans regia L.) and wheat (Triticum aestivum L.) have been widely applied in the Xinjiang Uygur Autonomous Region in Northwest China as a means of reducing soil and water losses and improving both land-use efficiency and economic returns. To understand how changes in soil conditions and nematode community structure can contribute to the evaluation of wheat–walnut intercropping systems from the view of soil fauna, we studied the soil nematode community in wheat rhizosphere soil under both monoculture and intercropping systems for 2 years. The results showed that the pH and total nitrogen and organic matter contents in intercropping systems with walnut trees were decreased compared with those of system with wheat alone. The nematode communities differed significantly between intercropping and monoculture plots, e.g., Rhabditis and Dorylaimus were dominant only in monocultures, whereas Tylenchus was dominant only in intercropping systems. Moreover, intercropping systems resulted in decreased nematode abundance, increased proportions of plant-feeding nematodes, and decreased omnivores/predators, particularly in the second year (2012). The decrease in diversity indices (H′) and ecological indices (WI, EI, and SI) of the nematode communities indicated high disturbance and low soil fertility in intercropping systems. Overall, wheat intercropping with walnut had a significant negative effect on wheat rhizosphere soil conditions.