Transcriptome analysis reveals key genes involved in the resistance to Cryphonectria parasitica during early disease development in Chinese chestnut.

BACKGROUND: Chestnut blight, one of the most serious branch diseases in Castanea caused by Cryphonectria parasitica, which has ravaged across American chestnut and most of European chestnut since the early twentieth century. Interestingly, the Chinese chestnut is strongly resistant to chestnut blight, shedding light on restoring the ecological status of Castanea plants severely affected by chestnut blight. To better explore the early defense of Chinese chestnut elicited in response to C. parasitica, the early stage of infection process of C. parasitica was observed and RNA sequencing-based transcriptomic profiling of responses of the chestnut blight-resistant wild resource 'HBY-1' at 0, 3 and 9 h after C. parasitica inoculation was performed. RESULTS: First, we found that 9 h was a critical period for Chinese chestnut infected by C. parasitica, which was the basis of further study on transcriptional activation of Chinese chestnut in response to chestnut blight in the early stage. In the transcriptome analysis, a total of 283 differentially expressed genes were identified between T9 h and Mock9 h, and these DEGs were mainly divided into two clusters, one of which was metabolism-related pathways including biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and photosynthesis; the other was related to plant-pathogen interaction and MAPK signal transduction. Meanwhile, the two clusters of pathways could be connected through junction among phosphatidylinositol signaling system, phytohormone signaling pathway and α-Linolenic acid metabolism pathway. It is worth noting that genes associated with JA biosynthesis and metabolic pathway were significantly up-regulated, revealing that the entire JA metabolic pathway was activated in Chinese chestnut at the early stage of chestnut blight infection. CONCLUSION: We identified the important infection nodes of C. parasitica and observed the morphological changes of Chinese chestnut wounds at the early stage of infection. In response to chestnut blight, the plant hormone and MAPK signal transduction pathways, plant-pathogen interaction pathways and metabolism-related pathways were activated at the early stage. JA biosynthesis and metabolic pathway may be particularly involved in the Chinese chestnut resistance to chestnut blight. These results contributes to verifying the key genes involved in the resistance of Chinese chestnut to C. parasitica.

Transcriptome analysis and characterization of genes associated to leaf tannin content in foxtail millet [Setaria italica (L.) P. Beauv.].

BACKGROUND: Chinese chestnut is an economically important tree species whose yield and quality are seriously affected by red spider attack. Tannins is one of the most important class secondary metabolites in plants, and is closely associated with plant defense mechanisms against insect and herbivory. In our previous studies, it was revealed that several low-tannin foxtail millet varieties growing under the Chinese chestnut trees could attract red spiders to feed on their leaves and protect the chestnut trees from the infestation of red spiders, meanwhile, the growth and yield of foxtail millet plants themselves were not greatly affected. RESULTS: To identify genes related to leaf tannin content and selection of foxtail millet germplasm resources with low tannin content for interplanting with Chinese chestnut and preventing the red spider attack, the leaves of 4 varieties with different levels of tannin content were harvested for comparative transcriptome analysis. In total, 335 differentially expressed genes (DEGs) were identified. For acquisition of gene functions and biological pathways they involved in, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed, and several DEGs were found to possibly participate in the tannins biosynthesis pathway and transport processes of precursors. In addition, according to the PlantTFDB database, some transcription factors were predicted among the DEGs, suggesting their role in regulation of tannins biosynthesis pathway. CONCLUSION: Our results provide valuable gene resources for understanding the biosynthesis and regulation mechanisms of tannins in foxtail millet, and pave the way for speeding up the breeding of low-tannin varieties through marker-assisted selection, which could be utilized for interplanting with Chinese chestnut trees to confer protection against red spider attack.

First report of Colletotrichum henanense causing anthracnose on Chinese chestnut in the United States.

Culinary chestnut production in the United States (US) is a rapidly growing industry supporting fresh market and value-added industries. An estimated 200-400 new acres of chestnuts are planted every year in the US, with most growers east of the Rocky Mountains planting Chinese chestnut (Castanea mollissima) or Chinese chestnut hybrids. In 2018, Ohio producers of Chinese chestnut reported losses of up to 80% to blossom end rot. Symptoms were like those reported by Fowler and Berry (1958), including black spots on the chestnut shell, often at the stylar end, and blackening of the kernels. Spots covered 1-100% of the kernel, however, no signs of any pathogen were present on the shell or kernel. In 2020, cankers that were brown/black in color, sunken, and ~1 cm in length were observed on 1-yr twigs of chestnut seedlings from a nursery operation on the same farm from which the symptomatic kernels were observed. In some seedlings, distal portions of the twigs died, while in other seedlings only shoots and leaves within the cankered areas died. Black acervuli were observed erupting from the cankers. Colletotrichum spp. were isolated and cultured on potato dextrose agar from surface-sterilized tissue from kernel lesions (MLI246-21 to MLI249-21) and twig cankers (MLI250-21 and MLI251-21). All isolates produced grey aerial mycelia, pink sporodochia, and cylindrical conidia with rounded ends ranging in size from 12-20 um long by 5-8 um wide. Isolates were preliminarily identified as belonging to the Colletotrichum gloeosporioides species complex (CGSC). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, ß-tubulin (TUB2) gene, and the intergenic spacer ApMat were amplified from genomic DNA and sequenced (Dowling et al. 2020). These genes are suitable for identifying species within the CGSC (Eaton et al. 2021). Sequences were submitted to the GenBank database (GAPDH OL687148 to OL687153, TUB2 OL741624 to OL741629 and ApMAT OL695914 to OL695919). BLASTn queries of NCBI GenBank showed that the GAPDH, TUB2, and ApMat sequences from all isolates had 98%, 98% and 99% identity with C. henanense isolates MT513015.1, MT513080.1, and MT512917.1 from apple (Martin et al. 2021). Representative isolates were used to demonstrate Koch's postulates and confirm pathogenicity on kernels (MLI246-21 and MLI249-21) and twigs (MLI250-21). Developing chestnuts in burs (n=4 per isolate) were gathered from the field and surface-sterilized with 70% ethanol. Nuts (n=12 per isolate) inside burs were inoculated by injecting 50uL of inoculum (~1.0 x 106 conidia/mL) directly into each kernel with a hypodermic needle and sterile syringe. Burs were incubated at room temperature in a moist chamber for 14 days. One-year-old seedlings (n=6) grown in containers were used for twig inoculations. Twig nodes (one/seedling) were surface sterilized with 70% ethanol and inoculated by wounding the stem with a sterile probe and dropping 100uL of inoculum into each wound. Seedlings were incubated in a glass house for 60 days and monitored daily for symptom development. Nuts (n=9) and twigs (n=3) were inoculated with sterile water using the same inoculation and incubation conditions to serve as negative controls. Inoculated kernels developed characteristic brown/black lesions at the wound site and inoculated twigs developed brown/black, slightly sunken cankers with acervuli. No symptoms developed on the control kernels or twigs. Fungi with the same colony, conidial, and molecular characteristics as C. henanense were re-isolated from inoculated kernels and twigs. Blossom end rot caused by Glomerella cingulata has been reported on chestnut kernels in Georgia (Fowler and Berry 1958), but this is the first report of C. henanense causing rot on kernels and twigs in the US. Since 2018, C. henanense has been isolated from infected nuts received from commercial orchards in Pennsylvania, Missouri, and Alabama. We propose to retire the name "blossom end rot" for the symptoms found in kernels and replace it with the name "chestnut anthracnose" for this disease that affects both twigs and kernels of Chinese chestnut.

Non-targeted and targeted metabolomics profiling of tea plants (Camellia sinensis) in response to its intercropping with Chinese chestnut.

BACKGROUND: Intercropping is often used in the tea producing areas where land resources are not so abundant, and the produced green tea is tasted more delicious through a tea-Chinese chestnut intercropping system according to the experience of indigenous farmers. The length and weight of tea leaf increase under this intercropping system and their root systems are stratified vertically and coordinate symbiosis. However, the delicacy mechanism under the intercropping is not fully understood. RESULTS: Green tea from the Chinese chestnut-tea intercropping system established in the 1980s ranked highest compared with a pure tea plantation from the same region. Based on the non-targeted metabolomics, 100 differential metabolites were upregulated in the tea leaves from intercropping system relative to monoculture system. Twenty-one amino acids were upregulated and three downregulated in response to the intercropping based on the targeted metabolomics; half of the upregulated amino acids had positive effects on the tea taste. Levels of allantoic acid, sugars, sugar alcohols, and oleic acid were higher and less bitter flavonoids in the intercropping system than those in monoculture system. The upregulated metabolites could promote the quality of tea and its health-beneficial health effects. Flavone and flavonol biosynthesis and phenylalanine metabolism showed the greatest difference. Numerous pathways associated with amino acid metabolism altered, suggesting that the intercropping of Chinese chestnut-tea could greatly influence amino acid metabolism in tea plants. CONCLUSIONS: These results enhance our understanding of the metabolic mechanisms by which tea quality is improved in the Chinese chestnut-tea intercropping system and demonstrate that there is great potential to improve tea quality at the metabolomic level by adopting such an intercropping system.

Transcriptome Analysis of Chinese Chestnut (Castanea mollissima Blume) in Response to Dryocosmus kuriphilus Yasumatsu Infestation.

Chinese chestnut (Castanea mollissima Blume) can be infested by Dryocosmus kuriphilus Yasumatsu, resulting in gall formation and yield losses. Research on the control of gall wasps using genomics approaches is rarely reported. We used RNA-seq to investigate the dynamic changes in the genes of a chestnut species (C. mollissima B.) during four gall-formation stages caused by D. kuriphilus. A total of 21,306 genes were annotated by BLAST in databases. Transcriptome comparison between different gall-formation stages revealed many genes that were differentially expressed compared to the control. Among these, 2410, 7373, 6294, and 9412 genes were differentially expressed in four gall-formation stages: initiation stage (A), early growth stage (B), late growth stage (C), and maturation stage (D), respectively. Annotation analysis indicated that many metabolic processes (e.g., phenylpropanoid biosynthesis, secondary metabolism, plant⁻pathogen interaction) were affected. Interesting genes encoding putative components of signal transduction, stress response, and transcription factors were also differentially regulated. These genes might play important roles in response to D. kuriphilus gall formation. These new data on the mechanism by which D. kuriphilus infests chestnuts could help improve chestnut resistance.

Comparison of Physicochemical Properties of Starches from Nine Chinese Chestnut Varieties.

Chestnut is a popular food in many countries and is also an important starch source. In previous studies, physicochemical properties of starches have been compared among different Chinese chestnut varieties growing under different conditions. In this study, nine Chinese chestnut varieties from the same farm were investigated for starch physicochemical properties to exclude the effects of growing conditions. The dry kernels had starch contents from 42.7 to 49.3%. Starches from different varieties had similar morphologies and exhibited round, oval, ellipsoidal, and polygonal shapes with a central hilum and smooth surface. Starch had bimodal size distribution and the volume-weighted mean diameter ranged from 7.2 to 8.2 µm among nine varieties. The starches had apparent amylose contents from 23.8 to 27.3% but exhibited the same C-type crystalline structure and similar relative crystallinity, ordered degree, and lamellar structure. The gelatinization onset, peak, and conclusion temperatures ranged from 60.4 to 63.9 °C, from 64.8 to 68.3 °C, and from 70.5 to 74.5 °C, respectively, among nine starches; and the peak, hot, breakdown, final, and setback viscosities ranged from 5524 to 6505 mPa s, from 3042 to 3616 mPa s, from 2205 to 2954 mPa s, from 4378 to 4942 mPa s, and from 1326 to 1788 mPa s, respectively. The rapidly digestible starch, slowly digestible starch, and resistant starch ranged from 2.6 to 3.7%, from 5.7 to 12.7%, and from 84.4 to 90.7%, respectively, for native starch, and from 79.6 to 89.5%, from 1.3 to 3.8%, and from 7.1 to 17.4%, respectively, for gelatinized starch.

Two new species of Dendrostoma (Erythrogloeaceae, Diaporthales) associated with Castaneamollissima canker disease in China.

The genus Dendrostoma is known to inhabit tree barks associated with branch canker diseases in China and several countries of Europe. Previous studies indicated that species of Dendrostoma prefer inhabiting fagaceous hosts, especially species of Castanea. In the present study, we obtained four isolates from cankered branches of Chinese chestnut (C.mollissima) in Rizhao City, Shandong Province, China. Morphological comparisons and phylogenetical analyses of a combined ITS-tef1-rpb2 sequence matrix were conducted, which revealed two new species named Dendrostomarizhaoense sp. nov. and D.tianii sp. nov. The new taxa are compared with other Dendrostoma species and comprehensive descriptions and illustrations are provided herein.

Metabolic and molecular mechanisms of spine color formation in Chinese red chestnut.

The spines of Chinese red chestnut are red and the depth of their color gradually increases with maturity. To identify the anthocyanin types and synthesis pathways in red chestnut and to identify the key genes regulating the anthocyanin biosynthesis pathway, we obtained and analyzed the transcriptome and anthocyanin metabolism of red chestnut and its control variety with green spines at 3 different periods. GO and KEGG analyses revealed that photosynthesis was more highly enriched in green spines compared with red spines, while processes related to defense and metabolism regulation were more highly enriched in red spines. The analysis showed that the change in spine color promoted photoprotection in red chestnut, especially at the early growth stage, which resulted in the accumulation of differentially expressed genes involved in the defense metabolic pathway. The metabolome results revealed 6 anthocyanins in red spines. Moreover, red spines exhibited high levels of cyanidin, peonidin and pelargonidin and low levels of delphinidin, petunidin and malvidin. Compared with those in the control group, the levels of cyanidin, peonidin, pelargonidin and malvidin in red spines were significantly increased, indicating that the cyanidin and pelargonidin pathways were enriched in the synthesis of anthocyanins in red spines, whereas the delphinidin pathways were inhibited and mostly transformed into malvidin. During the process of flower pigment synthesis, the expression of the CHS, CHI, F3H, CYP75A, CYP75B1, DFR and ANS genes clearly increased, that of CYP73A decreased obviously, and that of PAL, 4CL and LAR both increased and decreased. Notably, the findings revealed that the synthesized anthocyanin can be converted into anthocyanidin or epicatechin. In red spines, the upregulation of BZ1 gene expression increases the corresponding anthocyanidin content, and the upregulation of the ANR gene also promotes the conversion of anthocyanin to epicatechin. The transcription factors involved in color formation included 4 WRKYs.

Terpene Synthase Gene Family in Chinese Chestnut (Castanea mollissima BL.) Harbors Two Sesquiterpene Synthase Genes Implicated in Defense against Gall Wasp Dryocosmus kuriphilus.

Chinese chestnut (Castanea mollissima BL.) is a well-known fruit tree that has been cultivated in East Asia for millennia. Leaves and buds of the plant can become seriously infested by the gall wasp Dryocosmus kuriphilus (GWDK), which results in gall formation and associated significant losses in fruit production. Herbivore-induced terpenes have been reported to play an important role in plant-herbivory interactions, and in this study, we show that upon herbivory by GWDK, four terpene-related compounds were significantly induced, while the concentrations of these four compounds in intact buds were relatively low. Among these compounds, (E)-nerolidol and (E, E)-α-farnesene have frequently been reported to be involved in plant herbivory defenses, which suggests direct and/or indirect functions in chestnut GWDK defenses. Candidate terpene synthase (TPS) genes that may account for (E)-nerolidol and (E, E)-α-farnesene terpene biosynthesis were characterized by transcriptomics and phylogenetic approaches, which revealed altered transcript levels for two TPSs: CmAFS, a TPS-g subfamily member, and CmNES/AFS, a TPS-b clade member. Both genes were dramatically upregulated in gene expression upon GWDK infestation. Furthermore, Agrobacterium tumefaciens-mediated transient overexpression in Nicotiana benthamiana showed that CmAFS catalyzed the formation of (E, E)-α-farnesene, while CmNES/AFS showed dual (E)-nerolidol and (E, E)-α-farnesene synthase activity. Biochemical assays of the recombinant CmAFS and CmNES/AFS proteins confirmed their catalytic activity in vitro, and the enzymatic products were consistent with two of the major volatile compounds released upon GWDK-infested chestnut buds. Subcellular localization demonstrated that CmAFS and CmNES/AFS were both localized in the cytoplasm, the primary compartment for sesquiterpene synthesis. In summary, we show that two novel sesquiterpene synthase genes CmAFS and CmNES/AFS are inducible by herbivory and can account for the elevated accumulation of (E, E)-α-farnesene and (E)-nerolidol upon GWDK infestation and may be implicated in chestnut defense against GWDK herbivores.

A transcriptomic evaluation of the mechanism of programmed cell death of the replaceable bud in Chinese chestnut.

Previous studies suggest that the senescence and death of the replaceable bud of the Chinese chestnut cultivar (cv.) "Tima Zhenzhu" involves programmed cell death (PCD). However, the molecular network regulating replaceable bud PCD is poorly characterized. Here, we performed transcriptomic profiling on the chestnut cv. "Tima Zhenzhu" replaceable bud before (S20), during (S25), and after (S30) PCD to unravel the molecular mechanism underlying the PCD process. A total of 5,779, 9,867, and 2,674 differentially expressed genes (DEGs) were discovered upon comparison of S20 vs S25, S20 vs S30, and S25 vs S30, respectively. Approximately 6,137 DEGs common to at least two comparisons were selected for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to interrogate the main corresponding biological functions and pathways. GO analysis showed that these common DEGs could be divided into three functional categories, including 15 cellular components, 14 molecular functions, and 19 biological processes. KEGG analysis found that "plant hormone signal transduction" included 93 DEGs. Overall, 441 DEGs were identified as related to the process of PCD. Most of these were found to be genes associated with ethylene signaling, as well as the initiation and execution of various PCD processes.

Transcriptome analysis and identification of genes associated with leaf crude protein content in foxtail millet [Setaria italica (L.) P. Beauv.].

Introduction: Spruce spider mite is a primary insect pest of Chinese chestnut in China and seriously influences its yield and quality. However, the current management against this mite is costly and poorly effective. In previous research, we bred several foxtail millet materials for interplanting with chestnut tree, and found that they had high levels of crude protein (CP) in leaves and attracted spruce spider mite to feed on the leaves, thereby reducing chestnut damage. Methods: In this study, four foxtail millet varieties with significant differences in leaf crude protein content were used for high-throughput sequencing and identification of genes associated with leaf crude protein content. Gene enrichment analyses were carried out to comprehend the functions of these genes and the biological processes in which they are involved. In addition, transcription factors (TFs) were evaluated. Results: 435 differentially expressed genes (DEGs) were identified, suggesting their potential role in crude protein accumulation. Some differentially expressed genes were found to be associated with nitrogen metabolism and ubiquitin-mediated proteolysis pathways. Moreover, we identified 40 TF genes categorized into 11 transcription factor families. Discussion: Our findings represent an important resource that clarifies the mechanisms of accumulation and control of leaf crude protein in foxtail millet, and provide an opportunity for suppression of spruce spider mite attack on Chinese chestnut by interplanting with foxtail millet varieties with high concentrations of leaf crude protein.

Genetic Diversity and Population Structure of Chinese Chestnut (Castanea mollissima Blume) Cultivars Revealed by GBS Resequencing.

Chinese chestnut (Castanea mollissima Bl.) is one of the earliest domesticated and cultivated fruit trees, and it is widely distributed in China. Because of the high quality of its nuts and its high resistance to abiotic and biotic stresses, Chinese chestnut could be used to improve edible chestnut varieties worldwide. However, the unclear domestication history and highly complex genetic background of Chinese chestnut have prevented the efficiency of breeding efforts. To explore the genetic diversity and structure of Chinese chestnut populations and generate new insights that could aid chestnut breeding, heterozygosity statistics, molecular variance analysis, ADMIXTURE analysis, principal component analysis, and phylogenetic analysis were conducted to analyze single nucleotide polymorphism data from 185 Chinese chestnut landraces from five geographical regions in China via genotyping by sequencing. Results showed that the genetic diversity level of the five populations from different regions was relatively high, with an observed heterozygosity of 0.2796-0.3427. The genetic diversity level of the population in the mid-western regions was the highest, while the population north of the Yellow River was the lowest. Molecular variance analysis showed that the variation among different populations was only 2.07%, while the intra-group variation reached 97.93%. The Chinese chestnut samples could be divided into two groups: a northern and southern population, separated by the Yellow River; however, some samples from the southern population were genetically closer to samples from the northern population. We speculate that this might be related to the migration of humans during the Han dynasty due to the frequent wars that took place during this period, which might have led to the introduction of chestnut to southern regions. Some samples from Shandong Province and Beijing City were outliers that did not cluster with their respective groups, and this might be caused by the special geographical, political, and economic significance of these two regions. The findings of our study showed the complex genetic relationships among Chinese chestnut landraces and the high genetic diversity of these resources.

Transcriptomics and Antioxidant Analysis of Two Chinese Chestnut (Castanea mollissima BL.) Varieties Provides New Insights Into the Mechanisms of Resistance to Gall Wasp Dryocosmus kuriphilus Infestation.

Chinese chestnut is a popular fruit tree with a high nutritional value of its nuts, which can suffer from infestation by the chestnut gall wasp Dryocosmus kuriphilus (GWDK) that results in gall formation and resultant loss of production and profitability. The physiological and molecular mechanisms of GWDK resistance found in certain genotypes currently remains elusive. To gain new insights into this phenomenon, a series of RNA-Seq integrated with metabolomic profiling experiments were executed to investigate the chemical and transcriptional differences in response to GWDK infestation in two contrasting chestnut varieties grown in China (the susceptible "HongLi," HL and the partially resistant "Shuhe_Wuyingli," SW). Three time points were selected for comparison: The initiation stage (A), growth stage (B), and maturation stage (C). Results showed that concentrations of hydrogen peroxide (H2O2) and the activities of peroxidase (POD) and superoxide dismutase (SOD) enzyme were elevated in the resistant SW leaves compared with those in HL leaves at all three developmental stages, while catalase (CAT) and polyphenol oxidase (PPO) activities were mostly higher in HL leaves. RNA-Seq transcriptomic analyses of HL and SW leaves revealed that various metabolic pathways involved in GWDK stress responses, such as plant hormone signal transduction, MAPK signaling, and the peroxisome pathway, were enriched in the contrasting samples. Moreover, the weighted gene co-expression network analysis (WGCNA) of differentially expressed genes in the POD pathway combined with transcription factors (TFs) indicated that the expression of TF members of bHLH, WRKY, NAC, and MYB family positively correlated with POD pathway gene expression. The TFs CmbHLH130 (EVM0032437), CmWRKY31 (EVM0017000), CmNAC50 (EVM0000033), and CmPHL12 (EVM0007330) were identified as putative TFs that participate in the regulation of insect-induced plant enzyme activities in chestnut, which may contribute to GWDK resistance in SW. Expression levels of 8 random differentially expressed genes (DEGs) were furthermore selected to perform quantitative reverse transcription PCR (qRT-PCR) to validate the accuracy of the RNA-Seq-derived expression patterns. This study guides the functional analyses of further candidate genes and mechanisms important for GWDK resistance in chestnuts in the future as well as can help in identifying the master transcriptional regulators and important enzyme steps that support major insect defense pathways in chestnut.

Transcriptome analysis and mining of genes related to shade tolerance in foxtail millet (Setaria italica (L.) P. Beauv.).

A stereo interplanting system with foxtail millet beneath chestnut trees is an effective planting method to raise the utilization of land in chestnut orchards, increase yields and improve quality of chestnut nuts. Consequently, exploration of genes involved in shade tolerance response in foxtail millet and breeding shade-tolerant varieties have become urgent issues. In this study, RNA-seq of leaf samples from two shade-tolerant varieties and three shade-intolerant varieties of foxtail millet at the booting stage was performed. Comparisons between the varieties revealed that 70 genes were commonly differentially expressed. Moreover, the ratio of net photosynthetic rate under shaded environment to that under light environment could be used as an indicator of shade tolerance. Subsequently, weighted gene co-expression network analysis was employed to construct a co-expression network and modules were correlated with this ratio. A total of 375 genes were identified as potentially relevant to shade tolerance, among which nine genes were also present in the 70 differentially expressed genes, which implied that they were good candidates for genes involved in shade tolerance. Our results provide valuable resources for elucidation of the molecular mechanisms underlying shade tolerance and will contribute to breeding of shade-tolerant foxtail millet that are adapted to the shaded environment under chestnut trees.

Development of Chinese chestnut whiskey: yeast strains isolation, fermentation system optimization, and scale-up fermentation.

In this study, we used Chinese chestnut as the main raw material to develop a novel type of whiskey. First, 16 yeasts were isolated and identified for producing aroma using olfactory plate assay. Of these, we screened nine yeast strains based on their fermentation capacity, aroma profile, and sensory evaluation. The results demonstrated the combination of strains HN006 (Saccharomyces cerevisiae) and HN010 (Wickerhamomyces anomalus) provided satisfactory wine fermentation with an interesting flavor profile, as strain HN010 was highly aromatic and had elevated sensory scores with comparatively low ethanol yield, while strain HN006 had a poor flavor profile but produced the largest amount of ethanol. Subsequently, we co-cultured strains HN006 and HN010 to optimize the fermentation system. The results revealed the following optimum parameters: a mixed inoculum of 6% (v/v) at an HN006/HN010 ratio of 1:2 (v/v), a raw material ratio of 5:3:2 (chestnut: malt: glutinous rice), and yeast extract concentration of 6 g/L. Additionally, this fermentation system was successfully scaled-up to a 1000 L pilot-scale system. The results of this study showed that strains HN006 and HN010 could be used as alternatives for whiskey fermentation, as well as provided a generalized experimental scheme to assess other microorganisms.

Construction of Pseudomolecules for the Chinese Chestnut (Castanea mollissima) Genome.

The Chinese chestnut (Castanea mollissima Bl.) is a woody nut crop with a high ecological value. Although many cultivars have been selected from natural seedlings, elite lines with comprehensive agronomic traits and characters remain rare. To explore genetic resources with aid of whole genome sequence will play important roles in modern breeding programs for chestnut. In this study, we generated a high-quality C. mollissima genome assembly by combining 90× Pacific Biosciences long read and 170× high-throughput chromosome conformation capture data. The assembly was 688.93 Mb in total, with a contig N50 of 2.83 Mb. Most of the assembled sequences (99.75%) were anchored onto 12 chromosomes, and 97.07% of the assemblies were accurately anchored and oriented. A total of 33,638 protein-coding genes were predicted in the C. mollissima genome. Comparative genomic and transcriptomic analyses provided insights into the genes expressed in specific tissues, as well as those associated with burr development in the Chinese chestnut. This highly contiguous assembly of the C. mollissima genome provides a valuable resource for studies aiming at identifying and characterizing agronomical-important traits, and will aid the design of breeding strategies to develop more focused, faster, and predictable improvement programs.

Structural, thermal, and hydrolysis properties of large and small granules from C-type starches of four Chinese chestnut varieties.

Large and small granules were separated from C-type starches of four Chinese chestnut varieties growing in the same environment. They had similar amylose contents from 17.7% to 20.2% and showed C-type crystallinity. The large granules had relative crystallinity from 19.2% to 20.3%, ordered degree from 0.672 to 0.706, and lamellar peak intensity from 233.2 to 267.1, but small granules had relative crystallinity from 16.2% to 18.2%, ordered degree from 0.635 to 0.663, and lamellar peak intensity from 201.6 to 213.1. The gelatinization peak temperatures ranged from 62.6 to 65.7 °C in large granules but from 60.3 to 61.7 °C in small granules, and enthalpy variation did from 12.5 to 13.7 J/g in large granules but from 10.1 to 11.7 J/g in small granules. Both large and small granules showed biphasic hydrolysis. Though small granules had significantly higher hydrolysis rate than large granules, but they had similar total hydrolysis extent during whole hydrolysis. The granule size had significantly positive relationships with relative crystallinity, ordered degree, lamellar peak intensity, and gelatinization temperature and enthalpy variation, but was negatively correlated to hydrolysis rate. The principal component analysis was conducted to reveal the interrelationships among different starch properties and the variations among different starches.

[Characteristics of nitrogen transportation in Castanea mollissima forest around the Fushi Reservoir watershed in north Zhejiang Province, China.] / æµ™æ±Ÿèµ‹çŸ³æ°´åº“é›†æ°´åŒºæ¿æ —æž—åœŸå£¤æ°®ç´ è¿ç§»ç‰¹å¾.

In this study, through setting runoff plots in Chinese chestnut (Castanea mollissima) fo-rest, the runoff water samples were collected and tested from January to October, 2013. Combining with soil nitrogen data before and after the rainy season, we analyzed the characteristics of soil nitrogen transportation in chestnut forest around the Fushi Reservoir watershed in north Zhejiang Pro-vince, China. The results showed that among the 10 surface runoff events of Chinese chestnut fo-rest, there exhibited a significant difference in runoff water volume of each runoff plot, and the maximum amount was 0.51 m3. Nitrogen was the major element associated with nutrient loss in the study region. The concentrations of nitrate nitrogen (NO3--N), ammonium nitrogen (NH4+-N), total nitrogen (TN) andchemical oxygen demend (CODMn) in the runoff water ranged from 0.02-1.87, 0.04-3.53, 1.69-5.33 and 5.30-14.07 mg·L-1, respectively. The water quality indexes were greatly affected by the runoff volume, and the relationship between the amount of nitrogen loss and runoff could be well fitted by using a linear equation. The difference in nitrogen contents of the soil in the upper, middle and lower part of the runoff plots was evident both before and after the rainy season. Moreover, the nitrogen content increased with the increasing altitude, but this trend decreased with increasing soil depth. Comparison of the difference between the four forms of soil nitrogen (NO3--N, NH4+-N, TN and hydrolyzable nitrogen Hydro-N of soil) before and after the rainy season showed that there existed significant differences in Hydro-N and TN, and the average diffe-rence values were 20.21 and 307.49 mg·kg-1, respectively. In this area, there was a great risk of nitrogen loss with runoff, and the potential non-point source pollution in Fushi Reservoir should be concerned.

[Effects of conversion of evergreen broad-leaved forest to Chinese chestnut plantation on soil organic carbon pools.] / å¤©ç„¶å¸¸ç»¿é˜”å¶æž—æ”¹é€ ä¸ºæ¿æ —æž—å¯¹åœŸå£¤æœ‰æœºç¢³åº“çš„å½±å“.

Converting natural forests to plantations significantly affects the characteristics of soil organic carbon (C) pools, due to the changes of vegetation cover and management practices. In this paper, to investigate the effects of conversion from evergreen broad-leaved forest (EBF) to Chinese chestnut plantation (CP) on soil organic C pool, the soils from surface (0-20 cm) and subsurface (20-40 cm) layers were sampled from the above two forests (the CP was converted from the EBF and had been intensively managed for 10 years) in Lin'an City, Zhejiang Province. The soil orga-nic C storage, labile organic C pools, and other basic soil properties were determined. The chemical composition of soil organic C was determined by nuclear magnetic resonance (NMR) technique. Results showed that the soil organic C storage, water soluble organic C (WSOC), hot water soluble organic C (HWSOC), microbial biomass C (MBC) and readily oxidizable C (ROC) concentrations in the surface layer decreased by 19.7%, 34.4%, 25.8%, 30.4% and 25.2%, respectively, after the conversion from EBF to CP. Such values for the subsurface layers decreased by 13.5%, 38.4%, 19.8%, 34.1% and 22.2%, respectively. The O-alkyl C content, aromatic C content and aromaticity of soil organic C in the surface layer decreased signi-ficantly by the land-use conversion, while alkyl C content, carbonyl C content and alkyl C to O-alkyl C (A/O-A) ratio increased signi-ficantly. The O-alkyl C content of soil organic C in the subsurface layer decreased significantly by the land-use conversion, alkyl C content and A/O-A ratio increased significantly, and aromatic C content, carbonyl C content and aromaticity of soil organic C were not changed. In conclusion, conve-rting EBF to CP and subsequent intensive management significantly decreased the soil organic C sto-rage and labile C pool contents and altered the chemical composition of soil organic C.

Effect of cooking methods on nutritional quality and volatile compounds of Chinese chestnut (Castanea mollissima Blume).

This study aimed to evaluate the effects of different cooking methods on the content of important nutrients and volatiles in the fruit of Chinese chestnut. The nutritional compounds, including starch, water-soluble protein, free amino acids, reducing sugar, sucrose, organic acids and total flavonoids, of boiled, roasted and fried chestnuts were significantly (P<0.05) lower than those of fresh chestnuts after cooking, while the amylose, fat, crude protein and total polyphenol content varied slightly (P>0.05). L-Aspartic acid, L-glutamic acid and L-arginine were found to be the main reduced free amino acids in cooked chestnuts. The main aromatic compositions in fresh chestnuts were aldehydes and esters, while ketones, furfural and furan were formed in cooked chestnuts due to the Maillard reaction and degradation of saccharides, amino acids and lipids. Principle component analysis demonstrated that roasting and frying had a similar effect on the nutritional composition of chestnuts, which differed from that of the boiling process.