Transcriptional dynamics reveals the asymmetrical events underlying graft union formation in pecan (Carya illinoinensis).

Grafting is a widely used technique for pecan propagation; however, the background molecular events underlying grafting are still poorly understood. In our study, the graft partners during pecan [Carya illinoinensis (Wangenh.) K. Koch] graft union formation were separately sampled for RNA-seq, and the transcriptional dynamics were described via weighted gene co-expression network analysis. To reveal the main events underlying grafting, the correlations between modules and grafting traits were analyzed. Functional annotation showed that during the entire graft process, signal transduction was activated in the scion, while messenger RNA splicing was induced in the rootstock. At 2 days after grafting, the main processes occurring in the scion were associated with protein synthesis and processing, while the primary processes occurring in the rootstock were energy release-related. During the period of 7-14 days after grafting, defense response was a critical process taking place in the scion; however, the main process functioning in the rootstock was photosynthesis. From 22 to 32 days after grafting, the principal processes taking place in the scion were jasmonic acid biosynthesis and defense response, whereas the highly activated processes associated with the rootstock were auxin biosynthesis and plant-type secondary cell wall biogenesis. To further prove that the graft partners responded asymmetrically to stress, hydrogen peroxide contents as well as peroxidase and ß-1,3-glucanase activities were detected, and the results showed that their levels were increased in the scion not the rootstock at certain time points after grafting. Our study reveals that the scion and rootstock might respond asymmetrically to grafting in pecan, and the scion was likely associated with stress response, while the rootstock was probably involved in energy supply and xylem bridge differentiation during graft union formation.

Proteomic and Transcriptomic Analyses Provide New Insights into the Mechanism Underlying Lipid Deterioration in Pecan Kernels during Storage.

Pecan nuts are rich in lipids that tend to deteriorate during storage. Tandem mass-tag-based quantitative proteomics and transcriptomics were used to investigate the changes in the protein and gene profiles of stored pecan kernels for the first time. Our previous lipidomic data were jointly analyzed to elucidate the coordinated changes in lipid molecules and related proteins/genes. The mechanism underlying lipid deterioration in pecan kernels during storage was revealed by multiomics analyses. Lipid metabolism-related pathways were activated during pecan storage. Phospholipases, triacylglycerol lipases, lipoxygenases, and oil body-related proteins/genes were highly expressed during storage, revealing their involvement in lipid deterioration. These data provide rich information and will be valuable for future genetic or chemical research to alleviate lipid deterioration in pecans.

Transcriptome profile of pecan scab resistant and susceptible trees from a pecan provenance collection.

Pecan scab is a devastating disease that causes damage to pecan (Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab.The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples.Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Characteristic analysis of BZR genes family and their responses to hormone treatments and abiotic stresses in Carya illinoinensis.

As the core of Brassinosteroids (BR) signaling pathway, BR-resistant (BZR) transcription factor regulates thousands of targeted genes mediating photomophogenesis, pollen sterility, cell expansion and stress response. Pecan (Carya illinoinensis) is a famous trees species of Carya, and its nut has high nutritional and economic values. However, there has no report on BZR genes family in pecan yet. Herein, totals of seven CiBZR members were identified in pecan genome, which were predicted to be hydrophilic unstable proteins and located in the nucleus. CiBZR genes had close evolutionary relationships with CcBZRs and JrBZRs in both Carya cathayensis and Juglans regia. These seven CiBZR genes were located independently on 7 chromosomes without doubling or tandem duplication. Based on the analysis of conserved motifs and gene structures, CiBZR genes were divided into three categories. More than 40 cis-acting elements were found in the 2 kb promoter regions of CiBZRs, which were mainly involved in hormone, light, and stress response, and plant growth and development. Notably, some of these CiBZR proteins were mainly located in the nucleus, had the self-activation ability and interaction relationship with BIN2 kinase, and negatively regulated the expression of CiCPD and CiDWF4. Gene expressions analysis further showed that CiBZR genes could express in many tissues and shared similar expression trends during embryo development. Moreover, most CiBZR genes responded to BR, Gibberellin (GA), Strigolactone (SL), salt, acid and osmotic stress. This study provides theoretical basis for the subsequent study on the role of CiBZR family genes in plant growth, development and stress responses.

Old but still good: genetic diversity of ancient pecan genotypes from southern Brazil.

Pecan [Carya illinoinensis (Wangenh.) K. Koch] is a crop fruit native to the USA and Mexico currently cultivated in several countries, including Brazil, Uruguay, Argentina, Chile, Peru, China, South Africa, and Australia. Supported by the increasing consumption and market prices, the interest in the cultivation of this fruit crop is strongly growing around the world. In this study, AFLP and S-SAP markers were employed to characterize the genetic diversity of ancient accessions of pecan from southern Brazil. The evaluated plants were selected and preserved by the farmers and are remnants of the first introduction of seedlings from the U.S.A into southern Brazil aiming at developing research towards establishing commercial orchards. High levels of genetic diversity were estimated, suggesting that these plants have an important genetic background for the establishment of a germplasm collection with a wide genetic basis, for the development of breeding programs for this fruit crop. Cluster analysis of the genetic datasets revealed some correlation between the nuts' morphometric traits and genetic markers. Such correlation should be further exploited. These ancient genotypes must be evaluated for other agronomic traits of interest and included in core collections of pecans.

Genome-Wide Identification, Characterization, and Expression Analysis of Long-Chain Acyl-CoA Synthetases in Carya illinoinensis under Different Treatments.

As crucial enzymes in the lipid metabolic network, long-chain acyl-CoA synthases (LACSs) are members of the acyl-activated enzyme superfamily and play a crucial role in epidermal wax synthesis, plant lipid anabolic metabolism, and stress tolerance. In this study, 11 pecan LACS genes were identified and categorized into five groups and located on nine chromosomes. The significant degree of conservation in the AtLACS and CiLACS protein sequences was demonstrated by multiple sequence alignment and conserved motif analysis. Cis-acting element analysis identified numerous stress-responsive and hormone-inducible elements in the promoter regions of CiLACS genes. The expression levels of CiLACS9 and CiLACS9-1 were considerably up-regulated under salt and drought stress, according to the qRT-RCR study. Treatment with ABA also led to increased expression levels of CiLACS1, CiLACS1-1, CiLACS2, and CiLACS9-1. Notably, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9-1 exhibited peak expression levels at 135 days after anthesis and are likely to have been crucial in the accumulation of seed kernel oil. Moreover, the CiLACS9 gene was shown to be located in the cytoplasm. These findings offer a theoretical framework for clarifying the roles of LACS genes in the processes of pecan kernel oil synthesis and response to abiotic stressors.

Lipidomic and comparative transcriptomic analysis of fatty acid synthesis pathway in Carya illinoinensis embryo.

Pecan (Carya illinoinensis (Wagenh.) K. Koch) is an important oilseed nut and is rich in fatty acids (FAs) and flavonols. Pecan FA has significantly edible, industrial and clinical value. To investigate the dynamic patterns and compositions of FA, and the molecular mechanism that controls FA accumulation in pecan, lipidomic and transcriptomic analyses were performed to determine lipid profiles and gene expression in pecan's FA biosynthesis pathway. In the present study, compared with cultivars 'Caddo' and 'Y-01', 'Mahan' formed larger and heavier embryos and accumulated higher oil content. Lipidomic analysis showed that FA and (O-acyl)-1-hydroxy FA contents were higher in 'Mahan' at the mature stage. Based on full-length and comparative RNA-Seq, differential expression gene enrichment analysis revealed that many functional genes participated in the pathways of 'fatty acid biosynthesis', 'fatty acid metabolism' and 'linoleic acid metabolism'. High FA accumulation model from 'Mahan' demonstrated that key enzyme-encoding genes played an important role in regulating FA biosynthesis. Co-expression module analysis indicated that several transcription factors (TFs; MYB, TCP, bHLH, Dof, ERF, NAC) were involved in FA accumulation by regulating the expression of functional genes, and real-time quantitative PCR verification proved that these TFs had a high correlation with the pecan FA accumulation pattern. These findings provided an insight into the molecular mechanism of FA accumulation in C. illinoinensis embryo, which contributes to pecan oil yielding and pecan molecular breeding.

Differential Detection of Alternaria alternata Haplotypes Isolated from Carya illinoinensis Using PCR-RFLP Analysis of Alt a1 Gene Region.

Alternaria black spot disease on pecan is caused by the opportunistic pathogen Alternaria alternata and poses a serious threat to the local South African and global pecan industry. Several diagnostic molecular marker applications have been established and used in the screening of various fungal diseases worldwide. The present study investigated the potential for polymorphism within samples of A. alternata isolates obtained from eight different geographical locations in South Africa. Pecan (Carya illinoinensis) leaves, shoots, and nuts-in-shuck with Alternaria black spot disease were sampled, and 222 A. alternata isolates were retrieved. For rapid screening to identify Alternaria black spot pathogens, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the Alternaria major allergen (Alt a1) gene region was used, followed by the digestion of the amplicons with HaeIII and HinfI endonucleases. The assay resulted in five (HaeIII) and two (HinfI) band patterns. Unique banding patterns from the two endonucleases showed the best profile and isolates were grouped into six clusters using a UPGMA (unweighted pair group method with arithmetic averages) distance matrix (Euclidean) dendrogram method on R-Studio. The analysis confirmed that the genetic diversity of A. alternata does not depend on host tissues or the pecan cultivation region. The grouping of selected isolates was confirmed by DNA sequence analysis. The Alt a1 phylogeny corroborated no speciation within the dendrogram groups and showed 98-100% bootstrap similarity. This study reports the first documented rapid and reliable technique for routine screening identification of pathogens causing Alternaria black spot in South Africa.

WRINKLED1 Positively Regulates Oil Biosynthesis in Carya cathayensis.

Hickory (Carya cathayensis Sarg.) is a kind of important woody oil tree species, and its nut has high nutritional value. Previous gene coexpression analysis showed that WRINKLED1 (WRI1) may be a core regulator during embryo oil accumulation in hickory. However, its specific regulatory mechanism on hickory oil biosynthesis has not been investigated. Herein, two hickory orthologs of WRI1 (CcWRI1A and CcWRI1B) containing two AP2 domains with AW-box binding sites and three intrinsically disordered regions (IDRs) but lacking the PEST motif in the C-terminus were characterized. They are nucleus-located and have self-activated ability. The expression of these two genes was tissue-specific and relatively high in the developing embryo. Notably, CcWRI1A and CcWRI1B can restore the low oil content, shrinkage phenotype, composition of fatty acid, and expression of oil biosynthesis pathway genes of Arabidopsis wri1-1 mutant seeds. Additionally, CcWRI1A/B were shown to modulate the expression of some fatty acid biosynthesis genes in the transient expression system of nonseed tissues. Transcriptional activation analysis further indicated that CcWRI1s directly activated the expression of SUCROSE SYNTHASE2 (SUS2), PYRUVATE KINASE ß SUBUNIT 1 (PKP-ß1), and BIOTIN CARBOXYL CARRIER PROTEIN2 (BCCP2) involved in oil biosynthesis. These results suggest that CcWRI1s can promote oil synthesis by upregulating some late glycolysis- and fatty acid biosynthesis-related genes. This work reveals the positive function of CcWRI1s in oil accumulation and provides a potential target for improving plant oil by bioengineering technology.

Transcriptome Analysis of Resistant and Susceptible Pecan (Carya illinoinensis) Reveals the Mechanism of Resistance to Black Spot Disease (Colletotrichum fioriniae).

Pecan, Carya illinoinensis (Wangenh.) K. Koch, is an important dried fruit and woody oil tree species grown worldwide. With continuous expansion of pecan cultivation, the frequency and scope of diseases, especially black spot disease, are increasing, damaging trees and reducing yields. In this study, the key factors in resistance to black spot disease (Colletotrichum fioriniae) were investigated between the high-resistance pecan variety "Kanza" and the low-resistance variety "Mahan". Leaf anatomy and antioxidase activities confirmed much stronger resistance to black spot disease in "Kanza" than in "Mahan". Transcriptome analysis indicated that the increased expression of genes associated with defense response, oxidation-reduction, and catalytic activity was involved in disease resistance. A connection network identified a highly expressed hub gene CiFSD2 (CIL1242S0042), which might participate in redox reactions to affect disease resistance. Overexpression of CiFSD2 in tobacco inhibited enlargement of necrotic spots and increased disease resistance. Overall, the expression of differentially expressed genes differed in pecan varieties with different levels of resistance to C. fioriniae infection. In addition, the hub genes associated with black spot resistance were identified and the functions clarified. The in-depth understanding of resistance to black spot disease provides new insights for early screening of resistant varieties and molecular-assisted breeding in pecan.

Comparative transcriptome analyses reveal insights into catkin bloom patterns in pecan protogynous and protandrous cultivars.

In perennial plants such as pecan, once reproductive maturity is attained, there are genetic switches that are regulated and required for flower development year after year. Pecan trees are heterodichogamous with both pistillate and staminate flowers produced on the same tree. Therefore, defining genes exclusively responsible for pistillate inflorescence and staminate inflorescence (catkin) initiation is challenging at best. To understand these genetic switches and their timing, this study analyzed catkin bloom and gene expression of lateral buds collected from a protogynous (Wichita) and a protandrous (Western) pecan cultivar in summer, autumn and spring. Our data showed that pistillate flowers in the current season on the same shoot negatively impacted catkin production on the protogynous 'Wichita' cultivar. Whereas fruit production the previous year on 'Wichita' had a positive effect on catkin production on the same shoot the following year. However, fruiting the previous year nor current year pistillate flower production had no significant effect on catkin production on 'Western' (protandrous cultivar) cultivar. The RNA-Seq results present more significant differences between the fruiting and non-fruiting shoots of the 'Wichita' cultivar compared to the 'Western' cultivar, revealing the genetic signals likely responsible for catkin production. Our data presented here, indicates the genes showing expression for the initiation of both types of flowers the season before bloom.

Genome structure-based Juglandaceae phylogenies contradict alignment-based phylogenies and substitution rates vary with DNA repair genes.

In lineages of allopolyploid origin, sets of homoeologous chromosomes may coexist that differ in gene content and syntenic structure. Presence or absence of genes and microsynteny along chromosomal blocks can serve to differentiate subgenomes and to infer phylogenies. We here apply genome-structural data to infer relationships in an ancient allopolyploid lineage, the walnut family (Juglandaceae), by using seven chromosome-level genomes, two of them newly assembled. Microsynteny and gene-content analyses yield identical topologies that place Platycarya with Engelhardia as did a 1980s morphological-cladistic study. DNA-alignment-based topologies here and in numerous earlier studies instead group Platycarya with Carya and Juglans, perhaps misled by past hybridization. All available data support a hybrid origin of Juglandaceae from extinct or unsampled progenitors nested within, or sister to, Myricaceae. Rhoiptelea chiliantha, sister to all other Juglandaceae, contains proportionally more DNA repair genes and appears to evolve at a rate 2.6- to 3.5-times slower than the remaining species.

CcMYB12 Positively Regulates Flavonoid Accumulation during Fruit Development in Carya cathayensis and Has a Role in Abiotic Stress Responses.

Flavonoid, an important secondary metabolite in plants, is involved in many biological processes. Its synthesis originates from the phenylpropane metabolic pathway, and it is catalyzed by a series of enzymes. The flavonoid biosynthetic pathway is regulated by many transcription factors, among which MYB transcription factors are thought to be key regulators. Hickory (Carya cathayensis) is an economic forest tree species belonging to the Juglandaceae family, and its fruit is rich in flavonoids. The transcriptome of exocarp and seed of hickory has previously been sequenced and analyzed by our team, revealing that CcMYB12 (CCA0691S0036) may be an important regulator of flavonoid synthesis. However, the specific regulatory role of CcMYB12 in hickory has not been clarified. Through a genome-wide analysis, a total of 153 R2R3-MYB genes were identified in hickory, classified into 23 subclasses, of which CcMYB12 was located in Subclass 7. The R2R3-MYBs showed a differential expression with the development of hickory exocarp and seed, indicating that these genes may regulate fruit development and metabolite accumulation. The phylogenetic analysis showed that CcMYB12 is a flavonol regulator, and its expression trend is the same as or opposite to that of flavonol synthesis-related genes. Moreover, CcMYB12 was found to be localized in the nucleus and have self-activation ability. The dual-luciferase reporter assay demonstrated that CcMYB12 strongly bonded to and activated the promoters of CcC4H, CcCHS, CcCHI, and CcF3H, which are key genes of the flavonoid synthesis pathway. Overexpression of CcMYB12 in Arabidopsis thaliana could increase the content of total flavonoids and the expression of related genes, including PAL, C4H, CHS, F3H, F3'H, ANS, and DFR, in the flavonoid synthesis pathway. These results reveal that CcMYB12 may directly regulate the expression of flavonoid-related genes and promote flavonoid synthesis in hickory fruit. Notably, the expression level of CcMYB12 in hickory seedlings was significantly boosted under NaCl and PEG treatments, while it was significantly downregulated under acid stress, suggesting that CcMYB12 may participate in the response to abiotic stresses. The results could provide a basis for further elucidating the regulation network of flavonoid biosynthesis and lay a foundation for developing new varieties of hickory with high flavonoid content.

Genome-Wide Identification and Expression Analysis of AMT and NRT Gene Family in Pecan (Carya illinoinensis) Seedlings Revealed a Preference for NH4+-N.

Nitrogen (N) is a major limiting factor for plant growth and crop production. The use of N fertilizer in forestry production is increasing each year, but the loss is substantial. Mastering the regulatory mechanisms of N uptake and transport is a key way to improve plant nitrogen use efficiency (NUE). However, this has rarely been studied in pecans. In this study, 10 AMT and 69 NRT gene family members were identified and systematically analyzed from the whole pecan genome using a bioinformatics approach, and the expression patterns of AMT and NRT genes and the uptake characteristics of NH4+ and NO3- in pecan were analyzed by aeroponic cultivation at varying NH4+/NO3- ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that gene duplication was the main reason for the amplification of the AMT and NRT gene families in pecan, both of which experienced purifying selection. Based on qRT-PCR results, CiAMTs were primarily expressed in roots, and CiNRTs were majorly expressed in leaves, which were consistent with the distribution of pecan NH4+ and NO3- concentrations in the organs. The expression levels of CiAMTs and CiNRTs were mainly significantly upregulated under N deficiency and T4 treatment. Meanwhile, T4 treatment significantly increased the NH4+, NO3-, and NO2- concentrations as well as the Vmax and Km values of NH4+ and NO3- in pecans, and Vmax/Km indicated that pecan seedlings preferred to absorb NH4+. In summary, considering the single N source of T5, we suggested that the NH4+/NO3- ratio of 75:25 was more beneficial to improve the NUE of pecan, thus increasing pecan yield, which provides a theoretical basis for promoting the scale development of pecan and provides a basis for further identification of the functions of AMT and NRT genes in the N uptake and transport process of pecan.

Transcriptome Analysis Reveals a Comprehensive Virus Resistance Response Mechanism in Pecan Infected by a Novel Badnavirus Pecan Virus.

Pecan leaf-variegated plant, which was infected with a novel badnavirus named pecan mosaic virus (PMV) detected by small RNA deep sequencing, is a vital model plant for studying the molecular mechanism of retaining green or chlorosis of virus-infected leaves. In this report, PMV infection in pecan leaves induced PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). PMV infection suppressed the expressions of key genes of fatty acid, oleic acid (C18:1), and very-long-chain fatty acids (VLCFA) biosynthesis, indicating that fatty acids-derived signaling was one of the important defense pathways in response to PMV infection in pecan. PMV infection in pecans enhanced the expressions of pathogenesis-related protein 1 (PR1). However, the transcripts of phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) were downregulated, indicating that salicylic acid (SA) biosynthesis was blocked in pecan infected with PMV. Meanwhile, disruption of auxin signaling affected the activation of the jasmonic acid (JA) pathway. Thus, C18:1 and JA signals are involved in response to PMV infection in pecan. In PMV-infected yellow leaves, damaged chloroplast structure and activation of mitogen-activated protein kinase 3 (MPK3) inhibited photosynthesis. Cytokinin and SA biosynthesis was blocked, leading to plants losing immune responses and systemic acquired resistance (SAR). The repression of photosynthesis and the induction of sink metabolism in the infected tissue led to dramatic changes in carbohydrate partitioning. On the contrary, the green leaves of PMV infection in pecan plants had whole cell tissue structure and chloroplast clustering, establishing a strong antiviral immunity system. Cytokinin biosynthesis and signaling transductions were remarkably strengthened, activating plant immune responses. Meanwhile, cytokinin accumulation in green leaves induced partial SA biosynthesis and gained comparatively higher SAR compared to that of yellow leaves. Disturbance of the ribosome biogenesis might enhance the resistance to PMV infection in pecan and lead to leaves staying green.

Transcriptomic Analysis to Unravel Potential Pathways and Genes Involved in Pecan (Carya illinoinensis) Resistance to Pestalotiopsis microspora.

Fruit black spot (FBS), a fungal disease of pecan (Carya illinoinensis (Wangenh) K. Koch) caused by the pathogen Pestalotiopsis microspora, is a serious disease and poses a critical threat to pecan yield and quality. However, the details of pecan responses to FBS infection at the transcriptional level remain to be elucidated. In present study, we used RNA-Seq to analyze differential gene expression in three pecan cultivars with varied resistance to FBS infection: Xinxuan-4 (X4), Mahan (M), and Wichita (W), which were categorized as having low, mild, and high susceptibility to FBS, respectively. Nine RNA-Seq libraries were constructed, comprising a total of 58.56 Gb of high-quality bases, and 2420, 4380, and 8754 differentially expressed genes (DEGs) with |log2Fold change| ≥ 1 and p-value < 0.05 were identified between M vs. X4, W vs. M, and W vs. X4, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analyses were performed to further annotate DEGs that were part of specific pathways, which revealed that out of 134 total pathways, MAPK signaling pathway, plant−pathogen interaction, and plant hormone signal transduction were highly enriched. Transcriptomic profiling analysis revealed that 1681 pathogen-related genes (PRGs), including 24 genes encoding WRKY transcription factors, potentially participate in the process of defense against Pestalotiopsis microspora infection in pecan. The correlation of WRKY TFs and PRGs was also performed to reveal the potential interaction networks among disease-resistance/pathogenesis-related genes and WRKY TFs. Expression profiling of nine genes annotated as TIFY, WRKY TF, and disease-resistance protein-related genes was performed using qRT-PCR, and the results were correlated with RNA-Seq data. This study provides valuable information on the molecular basis of pecan−Pestalotiopsis microspora interaction mechanisms and offers a repertoire of candidate genes related to pecan fruit response to FBS infection.

Population Genetic Characteristics and Mating Type Frequency of Venturia effusa from Pecan in South America.

Scab, caused by the plant-pathogenic fungus Venturia effusa, is a major disease of pecan in South America, resulting in loss of quantity and quality of nut yield. Characteristics of the populations of V. effusa in South America are unknown. We used microsatellites to describe the genetic diversity and population structure of V. effusa in South America, and determined the mating type status of the pathogen. The four hierarchically sampled orchard populations from Argentina (AR), Brazil (BRC and BRS), and Uruguay (UR) had moderate to high genotypic and gene diversity. There was evidence of population differentiation (Fst = 0.196) but the correlation between geographic distance and genetic distance was not statistically significant. Genetic differentiation was minimal between the UR, BRC, and BRS populations, and these populations were more clearly differentiated from the AR population. The MAT1-1 and MAT1-2 mating types occurred in all four orchards and their frequencies did not deviate from the 1:1 ratio expected under random mating; however, multilocus linkage equilibrium was rejected in three of the four populations. The population genetics of South American populations of V. effusa has many similarities to the population genetics of V. effusa previously described in the United States. Characterizing the populations genetics and reproductive systems of V. effusa are important to establish the evolutionary potential of the pathogen and, thus, its adaptability-and can provide a basis for informed approaches to utilizing available host resistance and determining phytosanitary needs.

Genome-Wide Identification, Characterization, and Expression Profiling of AP2/ERF Superfamily Genes under Different Development and Abiotic Stress Conditions in Pecan (Carya illinoinensis).

The ethylene-responsive element (AP2/ERF) is one of the keys and conserved transcription factors (TFs) in plants that play a vital role in regulating plant growth, development, and stress response. A total of 202 AP2/ERF genes were identified from the pecan genome and renamed according to the chromosomal distribution of the CiAP2/ERF genes. They were divided into four subfamilies according to the domain and phylogenetic analysis, including 26 AP2, 168 ERF, six RAV, and two Soloist gene family members. These genes were distributed randomly across the 16 chromosomes, and we found 19 tandem and 146 segmental duplications which arose from ancient duplication events. The gene structure and conserved motif analysis demonstrated the conserved nature of intron/exon organization and motifs among the AP2/ERF genes. Several cis-regulatory elements, which were related to light responsiveness, stress, and defense responses, were identified in the promoter regions of AP2/ERFs. The expression profiling of 202 CiAP2/ERF genes was assessed by using RNA-Seq data and qRT-PCR during development (pistillate flowering development, graft union development, and kernel development) and under abiotic stresses (waterlogging, drought). Moreover, the results suggested that the ERF-VII members may play a critical role in waterlogging stress. These findings provided new insights into AP2/ERF gene evolution and divergence in pecan and can be considered a valuable resource for further functional validation, as well as for utilization in a stress-resistance-variety development program.

Integrated physiological, proteomic, and metabolomic analyses of pecan cultivar 'Pawnee' adaptation to salt stress.

The pecan is a salt-alkali-tolerant plant, and its fruit and wood have high economic value. This study aimed to explore the molecular mechanisms responsible for salt stress tolerance in the pecan grown under hydroponic conditions to simulate salt stress. The results showed that the photosynthetic rate (Pn) was reduced in response to salt stress, while the intercellular carbon dioxide concentrations (Ci) increased. The response of the pecan to salt stress was measured using iTRAQ (isobaric tags for relative or absolute quantitation) and LC/MS (liquid chromatography and mass spectrometry) non-targeted metabolomics technology. A total of 198 differentially expressed proteins (65 down-regulated and 133 up-regulated) and 538 differentially expressed metabolites (283 down-regulated and 255 up-regulated) were identified after exposure to salt stress for 48 h. These genes were associated with 21 core pathways, shown by Kyoto Encyclopedia of Genes and Genomes annotation and enrichment, including the metabolic pathways involved in nucleotide sugar and amino sugar metabolism, amino acid biosynthesis, starch and sucrose metabolism, and phenylpropane biosynthesis. In addition, analysis of interactions between the differentially expressed proteins and metabolites showed that two key nodes of the salt stress regulatory network, L-fucose and succinate, were up-regulated and down-regulated, respectively, suggesting that these metabolites may be significant for adaptations to salt stress. Finally, several key proteins were further verified by parallel reaction monitoring. In conclusion, this study used physiological, proteomic, and metabolomic methods to provide an important preliminary foundation for improving the salt tolerance of pecans.

The Complete Chloroplast Genome of Carya cathayensis and Phylogenetic Analysis.

Carya cathayensis, an important economic nut tree, is narrowly endemic to eastern China in the wild. The complete cp genome of C. cathayensis was sequenced with NGS using an Illumina HiSeq2500, analyzed, and compared to its closely related species. The cp genome is 160,825 bp in length with an overall GC content of 36.13%, presenting a quadripartite structure comprising a large single copy (LSC; 90,115 bp), a small single copy (SSC; 18,760 bp), and a pair of inverted repeats (IRs; 25,975 bp). The genome contains 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. A total of 252 simple sequence repeats (SSRs) and 55 long repeats were identified. Gene selective pressure analysis showed that seven genes (rps15, rpoA, rpoB, petD, ccsA, atpI, and ycf1-2) were possibly under positive selection compared with the other Juglandaceae species. Phylogenetic relationships of 46 species inferred that Juglandaceae is monophyletic, and that C. cathayensis is sister to Carya kweichowensis and Carya illinoinensis. The genome comparison revealed that there is a wide variability of the junction sites, and there is higher divergence in the noncoding regions than in coding regions. These results suggest a great potential in phylogenetic research. The newly characterized cp genome of C. cathayensis provides valuable information for further studies of this economically important species.