Genome-Wide Analysis of Transcription Factor R2R3-MYB Gene Family and Gene Expression Profiles during Anthocyanin Synthesis in Common Walnut (Juglans regia L.).

The R2R3-MYB gene family, encoding plant transcriptional regulators, participates in many metabolic pathways of plant physiology and development, including flavonoid metabolism and anthocyanin synthesis. This study proceeded as follows: the JrR2R3-MYB gene family was analyzed genome-wide, and the family members were identified and characterized using the high-quality walnut reference genome "Chandler 2.0". All 204 JrR2R3-MYBs were established and categorized into 30 subgroups via phylogenetic analysis. JrR2R3-MYBs were unevenly distributed over 16 chromosomes. Most JrR2R3-MYBs had similar structures and conservative motifs. The cis-acting elements exhibit multiple functions of JrR2R3-MYBs such as light response, metabolite response, and stress response. We found that the expansion of JrR2R3-MYBs was mainly caused by WGD or segmental duplication events. Ka/Ks analysis indicated that these genes were in a state of negative purifying selection. Transcriptome results suggested that JrR2R3-MYBs were widely entangled in the process of walnut organ development and differentially expressed in different colored varieties of walnuts. Subsequently, we identified 17 differentially expressed JrR2R3-MYBs, 9 of which may regulate anthocyanin biosynthesis based on the results of a phylogenetic analysis. These genes were present in greater expression levels in 'Zijing' leaves than in 'Lvling' leaves, as revealed by the results of qRT-PCR experiments. These results contributed to the elucidation of the functions of JrR2R3-MYBs in walnut coloration. Collectively, this work provides a foundation for exploring the functional characteristics of the JrR2R3-MYBs in walnuts and improving the nutritional value and appearance quality of walnuts.

Is winter coming? Minor effect of the onset of chilling accumulation on the prediction of endodormancy release and budbreak.

The buds of perennial plants become dormant in autumn and must integrate the information related to chilling and forcing temperatures to resume their growth in spring. In many studies, the initial date for chilling accumulation (DCA ) is set arbitrarily using various rules resulting in high variability across studies and sites. To test the relevancy of different rules to set DCA , sequential models (taking into account or not the negative effect of warm temperature) were optimized by minimizing the sums of squares between observed and predicted values for 34 endodormancy release and 77 budbreak dates for the walnut Juglans regia L. cv Franquette across France. Optimization of these different models highlighted that many of the DCA rules, incorporating a photoperiod signal on endodormancy induction, were effective (predicted root mean square standard error less than 10 and 8 days for endodormancy onset and bud break, respectively). Furthermore, the use of functions that compute negative chilling accumulation did not improve the performance of the models. Among the different rules, the projections of the best models were explored under different climates (current climate and Representative Concentration Pathways RCP scenarios). The projections revealed a tipping point at a mean annual temperature between 13 and 15°C, beyond which the advance in ontogenic development during ecodormancy does not compensate for the delay in endodormancy release. Although the physiological mechanisms driving the onset of endodormancy may be profoundly altered by global change, they appear to have minimal impact on the way current models predict dormancy and budbreak dates in walnut.

Screening and functional prediction of differentially expressed genes in walnut endocarp during hardening period based on deep neural network under agricultural internet of things.

The deep neural network is used to establish a neural network model to solve the problems of low accuracy and poor accuracy of traditional algorithms in screening differentially expressed genes and function prediction during the walnut endocarp hardening stage. The paper walnut is used as the research object to analyze the biological information of paper walnut. The changes of lignin deposition during endocarp hardening from 50 days to 90 days are observed by microscope. Then, the Convolutional Neural Network (CNN) and Long and Short-term Memory (LSTM) network model are adopted to construct an expression gene screening and function prediction model. Then, the transcriptome and proteome sequencing and biological information of walnut endocarp samples at 50, 57, 78, and 90 days after flowering are analyzed and taken as the training data set of the CNN + LSTM model. The experimental results demonstrate that the endocarp of paper walnut began to harden at 57 days, and the endocarp tissue on the hardened inner side also began to stain. This indicates that the endocarp hardened laterally from outside to inside. The screening and prediction results show that the CNN + LSTM model's highest accuracy can reach 0.9264. The Accuracy, Precision, Recall, and F1-score of the CNN + LSTM model are better than the traditional machine learning algorithm. Moreover, the Receiver Operating Curve (ROC) area enclosed by the CNN + LSTM model and coordinate axis is the largest, and the Area Under Curve (AUC) value is 0.9796. The comparison of ROC and AUC proves that the CNN + LSTM model is better than the traditional algorithm for screening differentially expressed genes and function prediction in the walnut endocarp hardening stage. Using deep learning to predict expressed genes' function accurately can reduce the breeding cost and significantly improve the yield and quality of crops. This research provides scientific guidance for the scientific breeding of paper walnut.

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.

Genome-Wide Identification of NAC Transcription Factor Family in Juglans mandshurica and Their Expression Analysis during the Fruit Development and Ripening.

The NAC (NAM, ATAF and CUC) gene family plays a crucial role in the transcriptional regulation of various biological processes and has been identified and characterized in multiple plant species. However, genome-wide identification of this gene family has not been implemented in Juglans mandshurica, and specific functions of these genes in the development of fruits remain unknown. In this study, we performed genome-wide identification and functional analysis of the NAC gene family during fruit development and identified a total of 114 JmNAC genes in the J. mandshurica genome. Chromosomal location analysis revealed that JmNAC genes were unevenly distributed in 16 chromosomes; the highest numbers were found in chromosomes 2 and 4. Furthermore, according to the homologues of JmNAC genes in Arabidopsis thaliana, a phylogenetic tree was constructed, and the results demonstrated 114 JmNAC genes, which were divided into eight subgroups. Four JmNAC gene pairs were identified as the result of tandem duplicates. Tissue-specific analysis of JmNAC genes during different developmental stages revealed that 39 and 25 JmNAC genes exhibited upregulation during the mature stage in walnut exocarp and embryos, indicating that they may serve key functions in fruit development. Furthermore, 12 upregulated JmNAC genes were common in fruit ripening stage in walnut exocarp and embryos, which demonstrated that these genes were positively correlated with fruit development in J. mandshurica. This study provides new insights into the regulatory functions of JmNAC genes during fruit development in J. mandshurica, thereby improving the understanding of characteristics and evolution of the JmNAC gene family.

Transcriptome profiling based on Illumina- and SMRT-based RNA-seq reveals circadian regulation of key pathways in flower bud development in walnut.

Flower bud development is a defining feature of walnut, which contributes to the kernel yield, yield stability, fruit quality and commodity value. However, little is known about the mechanism of the flower bud development in walnut. Here, the stages of walnut female flower bud development were divided into five period (P01-05) by using histological observation. They were further studied through PacBio Iso-Seq and RNA-seq analysis. Accordingly, we obtained 52,875 full-length transcripts, where 4,579 were new transcripts, 3,065 were novel genes, 1,437 were consensus lncRNAs and 20,813 were alternatively spliced isoforms. These transcripts greatly improved the current genome annotation and enhanced our understanding of the walnut transcriptome. Next, RNA sequencing of female flower buds at five periods revealed that circadian rhythm-plant was commonly enriched along with the flower bud developmental gradient. A total of 14 differentially expressed genes (DEGs) were identified, and six of them were confirmed by real-time quantitative analysis. Additionally, six and two differentially expressed clock genes were detected to be regulated by AS events and lncRNAs, respectively. All these detected plant circadian genes form a complex interconnected network to regulate the flower bud development. Thus, investigation of key genes associated with the circadian clock could clarify the process of flower bud development in walnut.

Antifungal Activity of Bacillus velezensis CE 100 against Anthracnose Disease (Colletotrichum gloeosporioides) and Growth Promotion of Walnut (Juglans regia L.) Trees.

Walnut anthracnose caused by Colletotrichum gloeosporioides is a deleterious disease that severely affects the production of walnut (Juglans regia L.). The aim of this study was to assess the antifungal and growth promotion activities of Bacillus velezensis CE 100 as an alternative to chemical use in walnut production. The crude enzyme from B. velezensis CE 100 exhibited chitinase, protease, and ß-l,3-glucanase activity and degraded the cell wall of C. gloeosporioides, causing the inhibition of spore germination and mycelial growth by 99.3% and 33.6% at 100 µL/mL, respectively. The field application of B. velezensis CE 100 culture broth resulted in a 1.3-fold and 6.9-fold decrease in anthracnose disease severity compared to the conventional and control groups, respectively. Moreover, B. velezensis CE 100 produced indole-3-acetic acid (up to 1.4 µg/mL) and exhibited the potential for ammonium production and phosphate solubilization to enhance the availability of essential nutrients. Thus, field inoculation of B. velezensis CE 100 improved walnut root development, increased nutrient uptake, enhanced chlorophyll content, and consequently improved total biomass by 1.5-fold and 2.0-fold compared to the conventional and control groups, respectively. These results demonstrate that B. velezensis CE 100 is an effective biocontrol agent against anthracnose disease and a potential plant growth-promoting bacteria in walnut tree production.

Effects of green-manure and tillage management on soil microbial community composition, nutrients and tree growth in a walnut orchard.

This study characterized the effect of green manures (February orchid, hairy vetch, rattail fescue and a no-green-manure control) and the termination method (flail or disk) on nutrient contents, enzyme activities, microbial biomass, microbial community structure of rhizosphere soil and vegetative growth of walnut tree. All three selected green manures significantly enhanced the water content, organic C, total N and available P. The rattail fescue significantly decreased the mineral N. Total organic C, total N, mineral N and available P were significantly greater under flail than under disk. Hairy vetch and February orchid significantly improved levels of soil ß-glucosidase, N-acetyl-glucosaminidase and acid phosphatase activity, whereas rattail fescue improved only ß-glucosidase activity. All of the green manures significantly decreased phenoloxidase activity. ß-glucosidase, N-acetyl-glucosaminidase and acid phosphatase activities were significantly greater under flail relative to disk. The termination method had no significant effect on phenoloxidase activity. The different types of green manures and termination methods significantly altered the soil microbial biomass and microbial community structure. The green-manure treatments were characterized by a significantly greater abundance of Gram-positive (Gram +) bacteria, total bacteria and saprophytic fungi compared to the control. Hairy vetch significantly decreased the abundance of arbuscular mycorrhizal fungi (AMF) while February orchid and rattail fescue increased their abundance compared to the no-green-manure treatment. The abundance rates of Gram+ bacteria, actinomycetes, saprophytic fungi and AMF were significantly greater in soils under flail than under disk. In terms of vegetative growth of walnut tree, hairy vetch showed the greatest positive effects. The growth of walnut tree was significantly greater under flail relative to disk. Our results indicate that green-manure application benefits the rhizosphere soil micro-ecology, rhizosphere soil nutrient contents and tree growth. Overall, the hairy vetch and flail combined treatment is recommended for walnut orchards in northern China.

The role of JrPPOs in the browning of walnut explants.

BACKGROUND: Tissue culture is an effective method for the rapid breeding of seedlings and improving production efficiency, but explant browning is a key limiting factor of walnut tissue culture. Specifically, the polymerization of PPO-derived quinones that cause explant browning of walnut is not well understood. This study investigated explants of 'Zanmei' walnut shoot apices cultured in agar (A) or vermiculite (V) media, and the survival percentage, changes in phenolic content, POD and PPO activity, and JrPPO expression in explants were studied to determine the role of PPO in the browning of walnut explants. RESULTS: The results showed that the V media greatly reduced the death rate of explants, and 89.9 and 38.7% of the explants cultured in V media and A media survived, respectively. Compared with that of explants at 0 h, the PPO of explants cultured in A was highly active throughout the culture, but activity in those cultured in V remained low. The phenolic level of explants cultured in A increased significantly at 72 h but subsequently declined, and the content in the explants cultured in V increased to a high level only at 144 h. The POD in explants cultured in V showed high activity that did not cause browning. Gene expression assays showed that the expression of JrPPO1 was downregulated in explants cultured in both A and V. However, the expression of JrPPO2 was upregulated in explants cultured in A throughout the culture and upregulated in V at 144 h. JrPPO expression analyses in different tissues showed that JrPPO1 was highly expressed in stems, young leaves, mature leaves, catkins, pistils, and hulls, and JrPPO2 was highly expressed in mature leaves and pistils. Moreover, browning assays showed that both explants in A and leaf tissue exhibited high JrPPO2 activity. CONCLUSION: The rapid increase in phenolic content caused the browning and death of explants. V media delayed the rapid accumulation of phenolic compounds in walnut explants in the short term, which significantly decreased explants mortality. The results suggest that JrPPO2 plays a key role in the oxidation of phenols in explants after branch injury.

Biocontrol and growth-promoting effect of Trichoderma asperellum TaspHu1 isolate from Juglans mandshurica rhizosphere soil.

To better apply the biocontrol agent Trichoderma spp. in Northeast China, collecting and screening more suitable native Trichoderma strains is necessary. In the present study, 10 isolates were obtained from Juglans mandshurica rhizosphere soils in Heilongjiang Province, and were identified as T. asperellum (four isolates), T. harzianum (four), T. hamatum (one), T. atroviride (one). The fastest-growing isolate per species on potato dextrose agar medium were further evaluated in stress tolerance tests (salt, alkali, nutritional stress, and low temperature) and confrontation assays (eight pathogens), which showed that T. asperellum TaspHu1 possessed the best adaptation and biological control ability. Then, Solanum lycopersicum (tomato) seeds were sown and treated with a series of concentrations of TaspHu1 spore suspension, as was unsown soil. Tomato seedlings treated by TaspHu1 had a significantly greater height, stem diameter, soluble protein content and soluble sugar content. Furthermore, their nitrate reductase activity and catalase activity were significantly increased, and these promoting effects depended on the concentration of the spore suspension. Meanwhile, a decrease in chlorophyll content was observed in the tomato seedlings treated with TaspHu1. In addition, strain TaspHu1 enhanced the tomato seedlings' absorption of available nitrogen, but did not influence the soil available nitrogen content. Furthermore, the resistance of tomato seedlings against Alternaria alternata was enhanced by TaspHu1 (smaller, fewer leaf spots), the seedlings' hormone signal transduction genes JAR1, MYC2, NPR1, PR1, and GH3.2 were highly expressed. Thus, TaspHu1 is a promising biocontrol candidate for use in agriculture and forestry.

Comparative transcriptome analyses provide novel insights into etiolated shoot development of walnut (Juglans regia L.).

MAIN CONCLUSION: In general, genes promoting IAA, CTK GA and ethylene biosynthesis were upregulated, while genes participating in ABA, chlorophyll and starch biosynthesis pathways performed opposite tendency during etiolation. Etiolation as a method for rejuvenation plays an important role in the vegetative propagation of woody plants. However, the molecular mechanism of etiolated shoot development remains unclear. In this study, we investigated changes at different etiolation stages of Juglans regia. The histology and transcriptome of J. regia were analysed using etiolated stems, which were treated in darkness for 30, 60, 90 days. The results showed that the ratios of pith (Pi) diameter/stem diameter (D), cortex (Co) width/D, and phloem (Ph) width/D increased, while the ratio of xylem (Xy) width/D decreased after etiolation, and the difference in these ratios between etiolated stems and the control was more significant at 60 days than 90 days. Differentially expressed genes (DEGs) were significantly enriched in pathways such as plant hormone biosynthesis and signal transduction, chlorophyll biosynthesis and degradation, and starch and sucrose metabolism. The difference in the contents of indole-3-acetic acid (IAA), abscisic acid (ABA), sugar and chlorophyll between etiolated stems and the control increased with increasing treatment duration; in contrast, the concentrations of gibberellin (GA), zeatin (ZT), and starch, as well as the difference between the etiolated stems and control were lowest at 60 days among the three stages. On the whole, the positive effect of etiolation on the rejuvenation of walnut stems changed as the treatment period increased. The present investigation lays a foundation for future studies on the effect of etiolation on rejuvenation and for promoting the efficiency of vegetative propagation.

Long-term effects of mixed planting on arbuscular mycorrhizal fungal communities in the roots and soils of Juglans mandshurica plantations.

BACKGROUND: Establishing mixed plantations is an effective way to improve soil fertility and increase forest productivity. Arbuscular mycorrhizal (AM) fungi are obligate symbiotic fungi that can promote mineral nutrient absorption and regulate intraspecific and interspecific competition in plants. However, the effects of mixed plantations on the community structure and abundance of AM fungi are still unclear. Illumina MiSeq sequencing was used to investigate the AM fungal community in the roots and soils of pure and mixed plantations (Juglans mandshurica × Larix gmelinii). The objective of this study is to compare the differential responses of the root and rhizosphere soil AM fungal communities of Juglans mandshurica to long-term mixed plantation management. RESULTS: Glomus and Paraglomus were the dominant genera in the root samples, accounting for more than 80% of the sequences. Compared with that in the pure plantation, the relative abundance of Glomus was higher in the mixed plantation. Glomus, Diversispora and Paraglomus accounted for more than 85% of the sequences in the soil samples. The relative abundances of Diversispora and an unidentified genus of Glomeromycetes were higher and lower in the pure plantation, respectively. The Root_P samples (the roots in the pure plantation) had the highest number of unique OTUs (operational taxonomic units), which belonged mainly to an unidentified genus of Glomeromycetes, Paraglomus, Glomus and Acaulospora. The number of unique OTUs detected in the soil was lower than that in the roots. In both the root and soil samples, the forest type did not have a significant effect on AM fungal diversity, but the Sobs value and the Shannon, Chao1 and Ace indices of AM fungi in the roots were significantly higher than those in the soil. CONCLUSIONS: Mixed forest management had little effect on the AM fungal community of Juglans mandshurica roots and significantly changed the community composition of the soil AM fungi, but not the diversity.

Evaluation of risk levels of trace elements in walnuts from China and their influence factors: Planting area and cultivar.

The potential health risk of trace elements in nut foods has been widely concerned. The accumulations of trace elements by plants in the environment are disturbed by multiple factors. The objective of this work was to investigate the risk levels of trace elements in walnuts and their influence factors (planting area and cultivar). A total of 228 walnut samples were collected from four major walnut producing areas of China. The contents of essential elements for Fe, Cu, Zn, Mo and Se were 35.8, 21.9, 14.8, 0.3 and 0.04 mg/kg, respectively. The contribution of Cu for dietary reference intake was as high as 82.22%. The risk levels of potential toxicological elements and toxic elements within the acceptable limits. Significantly, the hazard quotients (HQs) of Ba and Co were up to 26.14% and 25.31%, respectively. The effect of planting area on trace elements was determined from the aspects of regional distribution and urbanization. Significant differences of essential elements were found between northeast and northwest areas. The urbanization directly influenced toxic elements, which could cause variation up to 85.0% (Pb) and 42.9% (As). Finally, cultivar effect was confirmed that all walnut cultivars were divided into four categories with different trace element characteristics.

Flowering in Persian walnut: patterns of gene expression during flower development.

BACKGROUND: Flower development and sufficient fruit set are important parameters with respect to walnut yield. Knowledge about flowering genes of fruit trees can help to conduct better molecular breeding programs. Therefore, this study was carried out to investigate the expression pattern of some flowering genes (FT, SOC1, CAL, LFY and TFL1) in Persian walnut (cv. Chandler) during the growing season and winter dormancy. RESULTS: The results showed that walnut flower induction and initiation in Shahmirzad, Iran occurred in early June and late September, respectively. After meeting chilling and heat requirement, flower differentiation and anthesis occurred in late-March and mid-April to early-May, respectively. Study of flowering gene expression showed that the expression of the FT gene increased in three stages including before breaking of bud dormancy, from late March to late April (coincided with flower differentiation and anthesis) and from late May to mid-June (coincided with flower induction). Like FT, the expression of SOC1 gene increased during flower induction and initiation (mid-May to early-August) as well as flower anthesis (mid-April to early-May). LFY and CAL genes as floral meristem identity genes are activated by FT and SOC1 genes. In contrast with flowering stimulus genes, TFL1 showed overexpression during winter dormancy which prevented flowering. CONCLUSION: The expression of FT gene activated downstream floral meristem identity genes including SOC1, CAL and LFY which consequently led to release bud dormancy as well as flower anthesis and induction. Also, TFL1 as a flowering inhibitor gene in walnut showed overexpression during the bud dormancy. Chilling accumulation reduced TFL1 gene expression and increased the expression of flowering genes which ultimately led to overcome dormancy.

Animal wastes as fertilizers enhance growth of young walnut trees under soil drought conditions.

BACKGROUND: Using nutrient-rich animal wastes as organic fertilizers in agricultural practices is a sustainable method for soil amendment and avoiding environmental pollution. In order to evaluate their practical effect, we applied different proportions of animal waste as fertilizers to wet or dry soils that were either planted or not planted with young walnut trees. RESULTS: The results showed that animal waste could increase soil C accumulation and carbon to nitrogen (C/N) ratio and reduce soil organic nitrogen and total nitrogen contents as well as the nitrogen to phosphorus (N/P) ratio in the planted group soil. This framework of soil C and N composition (a high C/N ratio) resulted in high N and Mg contents as well as high Cu and Zn contents in the leaves of the young trees as well as a high dry matter weight/leaf N ratio, causing increased leaf photosynthesis, reduced transpiration and relatively high water use efficiency under soil drought conditions. Also, animal wastes as fertilizers caused the branching of walnut to switch from elongation growth to thickening growth under soil drought conditions. CONCLUSIONS: Principal component analysis and redundancy analysis demonstrated the mechanism by which the soil C/N ratio mediates the flux of available nutrients from the soil to the plant and thereby regulates plant dry matter accumulation and branching architecture under soil drought conditions. The results of this study provide new insights into the improvement of hilly soils using animal waste. © 2020 Society of Chemical Industry.

Comparison of phenological traits, growth patterns, and seasonal dynamics of non-structural carbohydrate in Mediterranean tree crop species.

Despite non-structural carbohydrate (NSC) importance for tree productivity and resilience, little is known about their seasonal regulations and trade-off with growth and reproduction. We characterize the seasonal dynamics of NSC in relation to the aboveground phenology and temporal growth patterns of three deciduous Mediterranean species: almond (Prunus dulcis (Mill.) D. A. Webb), walnut (Juglans regia L.) and pistachio (Pistacia vera L.). Seasonal dynamics of NSC were synchronous between wood tissues from trunk, branches and twigs. Almond had almost identical levels and patterns of NSC variation in twigs, branches and trunks whereas pistachio and walnut exhibited clear concentration differences among plant parts whereby twigs had the highest and most variable NSC concentration, followed by branches and then trunk. While phenology had a significant influence on NSC seasonal trends, there was no clear trade-off between NSC storage and growth suggesting that both were similarly strong sinks for NSC. A temporal trade-off observed at the seasonal scale was influenced by the phenology of the species. We propose that late senescing species experience C allocation trade-off at the end of the growing season because of C-limiting thermal conditions and priority allocation to storage in order to survive winter.

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.

Genome-wide Identification, Classification, Expression and Duplication Analysis of GRAS Family Genes in Juglans regia L.

Fifty-two GRAS genes are identified in walnut genome. Based on the evolutionary relationship and motif analysis, the walnut GRAS gene family was divided into eight subfamilies, and the sequence features analysis of JrGRAS proteins showed that the JrGRAS protein sequences were both conserved and altered during the evolutionary process. Gene duplication analysis indicated that seven GRAS genes in walnut have orthologous genes in other species, and five of them occurred duplicated events in walnut genome. Expression pattern analysis of the GRAS family genes in walnut showed that two JrGRAS genes (JrCIGRa-b and JrSCL28a) were differentially expressed between flower bud and leaf bud (p < 0.01), and two JrGRAS genes (JrCIGRa-b and JrSCL13b-d) were differentially expressed between the different development stages of flower buds transition (p < 0.01), besides, three hub genes (JrGAIa, JrSCL3f and JrSHRc) were identified by co-expression analysis, which suggested these GRAS genes may play an important role in regulating the development of apical meristem in walnut. This study laid a foundation for further understanding of the function of GRAS family genes in walnut.

Applying the AOGCM-AR5 models to the assessments of land suitability for walnut cultivation in response to climate change: A case study of Iran.

Due to higher temperatures and lower water availability, climate change is likely to have a major impact on walnut production in the near future. Climate change will alter the land suitability for walnut cultivation around the world, especially in arid and semi-arid regions like Iran. Here, land suitability for the cultivation of walnut (Juglans regia L.) in Iran was determined using the GIS for present and future conditions (2020-2049) with an approach to climate change. Accordingly, data from 375 synoptic stations throughout Iran were gathered for climatic factors including average, minimum and maximum temperatures, relative humidity and chilling requirement. Also, ASTER sensors (Advanced Spaceborne Thermal Emission and Reflection Radiometer) and their data provided this research with cells that make a precision of 150 m (5 s), and the data were used for gauging geological parameters such as altitude and land slope. The electrical conductivity (EC) of soil and water were informed by the data bank of the Iranian Water Resources Management. The results of temperature simulations for the future (2020-2049) were analyzed by 21 AOGCM-AR5 models under the RCP4.5 emission scenario. In the first phase of evaluations, the maps of land suitability were constructed for present conditions by considering a network of the above-mentioned parameters. By combining these layers of information, the final map of land suitability was illustrated for walnut cultivation. In the second phase, the NEX-GDDP was used in order to determine land suitability for the future (2020-2049). The results showed that Iran currently has 582844 km2 of land suitable for walnut cultivation. However, the future will see less suitable lands: the current area will be reduced by 6.19%, from 582844 km2 to 546710 km2. In general, the northern, northwestern and western margins of Iran are currently suitable for walnut cultivation. By approximation, these lands will also be major areas for prospective cultivations of walnut in the future (2020-2049), even though their current stretch will be reduced.

Stages identifying and transcriptome profiling of the floral transition in Juglans regia.

Using paraffin sections, the stages of walnut female flower bud differentiation were divided into the predifferentiation period (F_1), initial differentiation period (F_2) and flower primordium differentiation period (F_3). Leaf buds collected at the same stage as F_2 were designated JRL. Transcriptomic profiling was performed, and a total of 132,154 unigenes were obtained with lengths ranging from 201 bp to 16,831 bp. The analysis of differentially expressed genes (DEGs) showed that there were 597, 784 and 532 DEGs in the three combinations F_1vsF_2, F_1vsF_3, and F_2vsF_3, respectively. The comparison F_2vsJRL showed that 374 DEGs were differentially expressed between female buds and leaf buds. Thirty-one DEGs related to flowering time were further used to construct coexpression networks, and CRY2 and NF-YA were identified as core DEGs in flowering time regulation. Eighteen DEGs related to flowering time were subjected to real-time quantitative analysis. Our work provides a foundation for further research on the walnut floral transition and provides new resources for future research on walnut biology and biotechnology.