Identification and Characterization of Wall-Associated Kinase (WAK) and WAK-like (WAKL) Gene Family in Juglans regia and Its Wild Related Species Juglans mandshurica.

Wall-associated kinase (WAK) and WAK-like kinase (WAKL) are receptor-like kinases (RLKs), which play important roles in signal transduction between the cell wall and the cytoplasm in plants. WAK/WAKLs have been studied in many plants, but were rarely studied in the important economic walnut tree. In this study, 27 and 14 WAK/WAKL genes were identified in Juglans regia and its wild related species Juglans mandshurica, respectively. We found tandem duplication might play a critical role in the expansion of WAK/WAKL gene family in J. regia, and most of the WAK/WAKL homologous pairs underwent purified selection during evolution. All WAK/WAKL proteins have the extracellular WAK domain and the cytoplasmic protein kinase domain, and the latter was more conserved than the former. Cis-acting elements analysis showed that WAK/WAKL might be involved in plant growth and development, plant response to abiotic stress and hormones. Gene expression pattern analysis further indicated that most WAK/WAKL genes in J. regia might play a role in the development of leaves and be involved in plant response to biotic stress. Our study provides a new perspective for the evolutionary analysis of gene families in tree species and also provides potential candidate genes for studying WAK/WAKL gene function in walnuts.

Molecular and morphological evaluation of transgenic Persian walnut plants harboring Fld gene under osmotic stress condition.

BACKGROUND: Soil drought stress is a limiting factor of productivity in walnut (Juglans regia L). Ferredoxin (Fd) level decreases under adverse environmental stress. Functional replacement of decreased Fd by Fld (Flavodoxin) had been shown to have protective effect under abiotic stress condition. This study aimed to evaluate four transgenic lines (L3, L4, L13 and L17) along with non-transgenic line under three osmotic stresses levels (0, 10 and 12% PEG). METHODS AND RESULTS: This experiment carried out based on a completely randomized design with four replications. To confirm that the Fld gene is successfully integrated into the walnut genome, PCR and dot blot analysis were carried out. The transgenic lines of walnut expressing Fld displayed increased tolerance to osmotic stress at 10 and 12% PEG condition. Lines expressing Fld exhibited increasing tolerance to drought stress and maintained health of plants under osmotic conditions. Results of real time PCR showed that expression level of Fld gene in L4 was higher than the others. Among transgenic lines, L4 was more tolerant than other lines under osmotic stress. CONCLUSIONS: These findings indicate that expression of Fld gene can increase tolerance to osmotic stress in Persian walnut and is useful tool for walnut production in arid and semi-arid regions.

Authentication and Provenance of Walnut Combining Fourier Transform Mid-Infrared Spectroscopy with Machine Learning Algorithms.

Different varieties and geographical origins of walnut usually lead to different nutritional values, contributing to a big difference in the final price. The conventional analytical techniques have some unavoidable limitations, e.g., chemical analysis is usually time-expensive and labor-intensive. Therefore, this work aims to apply Fourier transform mid-infrared spectroscopy coupled with machine learning algorithms for the rapid and accurate classification of walnut species that originated from ten varieties produced from four provinces. Three types of models were developed by using five machine learning classifiers to (1) differentiate four geographical origins; (2) identify varieties produced from the same origin; and (3) classify all 10 varieties from four origins. Prior to modeling, the wavelet transform algorithm was used to smooth and denoise the spectrum. The results showed that the identification of varieties under the same origin performed the best (i.e., accuracy = 100% for some origins), followed by the classification of four different origins (i.e., accuracy = 96.97%), while the discrimination of all 10 varieties is the least desirable (i.e., accuracy = 87.88%). Our results implicated that using the full spectral range of 700-4350 cm-1 is inferior to using the subsets of the optimal spectral variables for some classifiers. Additionally, it is demonstrated that back propagation neural network (BPNN) delivered the best model performance, while random forests (RF) produced the worst outcome. Hence, this work showed that the authentication and provenance of walnut can be realized effectively based on Fourier transform mid-infrared spectroscopy combined with machine learning algorithms.

Chromosomal-level assembly of Juglans sigillata genome using Nanopore, BioNano, and Hi-C analysis.

BACKGROUND: Juglans sigillata, or iron walnut, belonging to the order Juglandales, is an economically important tree species in Asia, especially in the Yunnan province of China. However, little research has been conducted on J. sigillata at the molecular level, which hinders understanding of its evolution, speciation, and synthesis of secondary metabolites, as well as its wide adaptability to its plateau environment. To address these issues, a high-quality reference genome of J. sigillata would be useful. FINDINGS: To construct a high-quality reference genome for J. sigillata, we first generated 38.0 Gb short reads and 66.31 Gb long reads using Illumina and Nanopore sequencing platforms, respectively. The sequencing data were assembled into a 536.50-Mb genome assembly with a contig N50 length of 4.31 Mb. Additionally, we applied BioNano technology to identify contacts among contigs, which were then used to assemble contigs into scaffolds, resulting in a genome assembly with scaffold N50 length of 16.43 Mb and contig N50 length of 4.34 Mb. To obtain a chromosome-level genome assembly, we constructed 1 Hi-C library and sequenced 79.97 Gb raw reads using the Illumina HiSeq platform. We anchored ∼93% of the scaffold sequences into 16 chromosomes and evaluated the quality of our assembly using the high contact frequency heat map. Repetitive elements account for 50.06% of the genome, and 30,387 protein-coding genes were predicted from the genome, of which 99.8% have been functionally annotated. The genome-wide phylogenetic tree indicated an estimated divergence time between J. sigillata and Juglans regia of 49 million years ago on the basis of single-copy orthologous genes. CONCLUSIONS: We provide the first chromosome-level genome for J. sigillata. It will lay a valuable foundation for future research on the genetic improvement of J. sigillata.

The walnut genetic resources of INRA: chronological phenotypic data and ontology.

OBJECTIVES: Persian walnut (Juglans regia L.), the walnut species cultivated for nut production, is grown worldwide in temperate areas. In this work, chronological phenotypic data have been collected regarding a part of the walnut genetic resources of the French National Institute for Agricultural Research (INRA) of Bordeaux. Using a well described ontology, these data have been collected in order to assess the phenotypic variations among the accessions, and to better manage the germplasm collection. These data can also be helpful for any breeding program as they provide a clear phenotypic characterization of the main cultivars. DATA DESCRIPTION: This paper introduces a dataset collected for 150 J. regia accessions for a period from 1965 to 2016, and for 3 observation sites, released as comma separated value spreadsheet. It includes observations about phenological traits (e.g. flowering dates), traits related to in-shell walnut (e.g. weight and size), and traits related to kernel (e.g. color). It can be used by other researchers particularly for multi-site phenological studies in the context of climate change since climate data files are also available. In addition, a complete walnut ontology was deposited in this repository and can assist to standardize the management of any walnut germplasm collection.

Genomics: cracking the mysteries of walnuts.

The Juglans plants are economically important as they provide nuts, wood and garden trees. They also play an important ecological role by supplying food for wild insects and animals. The decoding of genome sequences has fundamental values for understanding the evolution of Juglans plants and molecules, and is also a prerequisite for molecular breeding. During the last three years, the rapid development of sequencing technology has made walnut research into the genome era. Here, we reviewed the progress of genome sequencing of six Juglans species, the resequencing of four Juglans populations as well as the genome sequencing of the closely related species Pterocarya stenoptera. The analysis of the Juglans regia genome uncovers a whole genome duplication (WGD) event. Based on the molecular dating of the divergence time of six Juglans species, we proposed this WGD event was associated with Cretaceous-Palaeogene (K-Pg) boundary occurred ∼65 million years ago. Genomic sequences also provide clear details for understanding the evolution and development of GGT and PPO genes involved in fruit development. The decoding of these genomes has made it easier for us to understand and enhance the use of walnuts. We expect that the functional genomics research of walnut will also develop rapidly in the near future.

¹H-NMR Profiling and Carbon Isotope Discrimination as Tools for the Comparative Assessment of Walnut (Juglans regia L.) Cultivars with Various Geographical and Genetic Origins-A Preliminary Study.

：The aim of the study was to investigate the differences between walnut genotypes of various geographical and genetic origins grown under the same or different environmental conditions. The biological material analyzed consisted in walnut kernels of 34 cultivars, nine advanced selections, and six hybrids harvested in 2015 and 2016, summing up to a total of 64 samples. The walnuts, walnut oil, and residue were characterized in respect to their chemical (proximate composition-fat, protein, nutritional value, fatty acids profile by ¹H-NMR) and carbon-13 isotopic composition. The data was used to statistically discriminate the cultivars according to composition, geographical area of origin, and year of harvest, comparing the Romanian cultivars, selections, and hybrids with the internationally available ones.

Genome-Wide Identification and Transcriptional Expression of the PAL Gene Family in Common Walnut (Juglans Regia L.).

Juglans regia L. is an economically important crop cultivated worldwide for its high quality and quantity of wood and nuts. Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway that plays a critical role in plant growth, development, and adaptation, but there have been few reports of the PAL gene family in common walnut. Here, we report a genome-wide study of J. regiaPAL genes and analyze their phylogeny, duplication, microRNA, and transcriptional expression. A total of 12 PAL genes were identified in the common walnut and clustered into two subfamilies based on phylogenetic analysis. These common walnut PALs are distributed on eight different pseudo-chromosomes. Seven of the 12 PALs (JrPAL2-3, JrPAL4-2, JrPAL2-1, JrPAL4-1, JrPAL8, JrPAL9, and JrPAL6) were specific found in J. regia, and JrPAL3, JrPAL5, JrPAL1-2, JrPAL7, and JrPAL2-2 were found to be closely associated with the woody plant Populus trichocarpa. Additionally, the expression patterns of JrPAL3, JrPAL7, JrPAL9, and JrPAL2-1 showed that they had high expression in female and male flowers. The miRNA ath-miR830-5p regulates two genes, JrPAL5 and JrPAL1, such that they have low expression in the male and female flowers of the common walnut. Our research provides useful information for further research into the function of PAL genes in common walnut and Juglans.

Genomic Variation Among and Within Six Juglans Species.

Genomic analysis in Juglans (walnuts) is expected to transform the breeding and agricultural production of both nuts and lumber. To that end, we report here the determination of reference sequences for six additional relatives of Juglans regia: Juglans sigillata (also from section Dioscaryon), Juglans nigra, Juglans microcarpa, Juglans hindsii (from section Rhysocaryon), Juglans cathayensis (from section Cardiocaryon), and the closely related Pterocarya stenoptera While these are 'draft' genomes, ranging in size between 640Mbp and 990Mbp, their contiguities and accuracies can support powerful annotations of genomic variation that are often the foundation of new avenues of research and breeding. We annotated nucleotide divergence and synteny by creating complete pairwise alignments of each reference genome to the remaining six. In addition, we have re-sequenced a sample of accessions from four Juglans species (including regia). The variation discovered in these surveys comprises a critical resource for experimentation and breeding, as well as a solid complementary annotation. To demonstrate the potential of these resources the structural and sequence variation in and around the polyphenol oxidase loci, PPO1 and PPO2 were investigated. As reported for other seed crops variation in this gene is implicated in the domestication of walnuts. The apparently Juglandaceae specific PPO1 duplicate shows accelerated divergence and an excess of amino acid replacement on the lineage leading to accessions of the domesticated nut crop species, Juglans regia and sigillata.

Characterization of virgin walnut oils and their residual cakes produced from different varieties.

This study addresses the composition and properties of different walnut varieties (Chandler, Hartley and Lara), in particular their virgin oils and residual cakes obtained by screw pressing employing different cultivars. Among nuts, walnut (Juglans regia L.) exhibits interesting nutritional value, mainly due to their high content in linoleic acid, phenolic and tocopherol compounds, which show antioxidant and other healthy properties. Valuable results related to fatty acid profile and minor components were observed. Virgin walnut oil is a rich source in linoleic acid (60-62%) and γ-tocopherol (517-554 mg/kg). Moreover, walnuts show a very high content in total phenolic compounds (10,045-12,474 mg/kg; as gallic acid), which contribute to a great antioxidant activity (105-170 mmol/kg for DPPH, and 260-393 mmol/kg for ORAC), being the hydrolysable tannins (2132-4204 mg/kg) and flavanols (796-2433 mg/kg) their main phenolic groups. Aldehydes account for the highest contribution to aromatic volatiles in virgin walnut oil (about 35% of total). As expected, polar phenolic compounds concentrate in the residual cake, after the separation of the oily phase, reaching a content of up to 19,869 mg/kg, leading to potential added value and applications as source of bioactive compounds to this by-product.

Phytochemical Study of Juglans regia L. Pericarps from Greece with a Chemotaxonomic Approach.

Phytochemical research of different polarity extracts from green Juglans regia L. pericarps from Greece afforded 32 compounds: four pentacyclic triterpenes (1 - 4), three sesquiterpenes (5 - 7), four tetralones (8 - 11), two naphthoquinones (12 and 13), seven phenolic acids (14 - 20), one diarylheptanoid (21), one neo-lignan (22), seven flavonoids (23 - 29), two phenylethanoids (30 and 31) and one hydrolysed tannin (32). Compounds 4 and 29 are isolated for the first time from the species, while compounds 3, 7, 20, 22, 23, 24, 25, 26, 28, 30 are reported for the first time in Juglandaceae. Chemotaxonomic significance of isolated compounds into Junglandaceae family is thoroughly discussed.

Phylogeography of postglacial range expansion in Juglans mandshurica (Juglandaceae) reveals no evidence of bottleneck, loss of genetic diversity, or isolation by distance in the leading-edge populations.

The past studies of postglacial recolonization patterns in high latitude regions have revealed a significant role of dispersal capacity in shaping the genetic diversity and population structure of temperate trees. However, most of these studies have focused on species with long-distance dispersal followed by exponential population growth and were therefore unable to reveal the patterns in the case of a gradual expansion. Here we studied the impacts of postglacial range expansions on the distribution of genetic diversity in the Manchurian walnut (Juglans mandshurica), a common tree of East Asian cool-temperate deciduous forests that apparently lacks long-distance seed dispersal ability. The genetic diversity and structure of 19 natural walnut populations in Northeast China and the Korean Peninsula were examined using 17 nuclear simple sequence repeat (SSR) loci. Potential habitats under current and past climatic conditions were predicted using the ecological niche modelling (ENM) method. Bayesian clustering analysis revealed three groups, which were inferred to have diverged through multiple glacial-interglacial cycles in multiple refugia during the Quaternary Period. ENM estimated a southward range shift at the LGM, but high suitability scores still occurred in the western parts of the Changbai Mountains (Northeast China), the Korean peninsula and the exposed seafloor of the Yellow Sea. In contrast to most other cool-temperate trees co-occurring in the same region, the Manchurian walnut did not show any evidence of a population bottleneck, loss of genetic diversity or isolation by distance during the postglacial expansion. Our study clearly indicates that current northern populations originated from one glacial lineage and recolonization via a gradually advancing front due to the lack of a long-distance seed dispersal mechanism led to no latitudinal decrease in genetic diversity.

[Cold-tolerance of walnut cultivars: a comprehensive evaluation].

In order to speed up the cold-tolerance breeding and improve the cultivation efficiency of walnut to further expand its planting area, six walnut cultivars (lines) including Luguo No. 8, N13-1, Luguo No. 12, N17-24, Taile, and Xiangling were taken as test materials and treated at -15 degrees C, -20 degrees C, -25 degrees C, -30 degrees C, and -35 degrees C, with their tissue's water content, membrane permeability, lipid peroxidation, protective enzyme activities, and osmotic adjustment substances determined to detect the cold resistance of the cultivars (lines). There existed greater differences in the ratio of free water to bound water in the branches of the cultivars (lines). Under low temperature treatment, the branches membrane permeability and malondialdehyde content increased, superoxide dismutase activity presented an ascend-descend-ascend-descend trend, catalase activity decreased after an initial increase, and the soluble protein and proline contents increased, but the variation degrees of these parameters differed with cultivars (lines). After the recovery from low temperature, N17-24 and Luguo No. 12 had a significantly higher germination rate than the other cultivars (lines). By using fuzzy mathematics method, the cold-resistance of test walnut cultivars (lines) was evaluated as N17-24 > Luguo No. 12 > N13-1 > Luguo No. 8 > Taile > Xiangling.

Total phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.) green husks.

The total phenols content and antioxidant and antimicrobial activities were studied in walnut (Juglans regia L.) green husks aqueous extracts of five different cultivars (Franquette, Mayette, Marbot, Mellanaise and Parisienne). Total phenols content was determined by colorimetric assay and their amount ranged from 32.61 mg/g of GAE (cv. Mellanaise) to 74.08 mg/g of GAE t (cv. Franquette). The antioxidant capacity of aqueous extracts was assessed through reducing power assay, scavenging effects on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and beta-carotene linoleate model system. A concentration-dependent antioxidative capacity was verified in reducing power and DPPH assays, with EC50 values lower than 1 mg/mL for all the tested extracts. The antimicrobial capacity was screened against Gram positive and Gram negative bacteria, and fungi. All the extracts inhibited the growth of Gram positive bacteria, being Staphylococcus aureus the most susceptible one with MIC of 0.1 mg/mL for all the extracts. The results obtained indicate that walnut green husks may become important in the obtainment of a noticeable source of compounds with health protective potential and antimicrobial activity.

Walnut (Juglans regia L.) leaves: phenolic compounds, antibacterial activity and antioxidant potential of different cultivars.

Different cultivars of walnut (Juglans regia L.) leaves (Cv. Lara, Franquette, Mayette, Marbot, Mellanaise and Parisienne) grown in Portugal, were investigated in what concerns phenolic compounds and antimicrobial and antioxidant properties. Phenolics analysis was performed by reversed-phase HPLC/DAD and 10 compounds were identified and quantified: 3- and 5-caffeoylquinic acids, 3- and 4-p-coumaroylquinic acids, p-coumaric acid, quercetin 3-galactoside, quercetin 3-pentoside derivative, quercetin 3-arabinoside, quercetin 3-xyloside and quercetin 3-rhamnoside. The antimicrobial capacity was screened against Gram positive (Bacillus cereus, B. subtilis, Staphylococcus aureus) and Gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) and fungi (Candida albicans, Cryptococcus neoformans). Walnut leaves selectively inhibited the growth of Gram positive bacteria, being B. cereus the most susceptible one (MIC 0.1mg/mL). Gram negative bacteria and fungi were resistant to the extracts at 100mg/mL. Lara walnut leaves were also submitted to antibacterial assays using 18 clinical isolates of Staphylococcus sp. Antioxidant activity was accessed by the reducing power assay, the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and beta-carotene linoleate model system. In a general way, all of the studied walnut leaves cultivars presented high antioxidant activity (EC(50) values lower than 1mg/mL), being Cv. Lara the most effective one.