WRKY transcription factors play an important role in the regulation of plant growth and defence responses.In order to explore the function of WRKY gene family in chestnut under drought resistance,the chestnut WRKY gene family members were identified and the physical and chemical properties,phylogeny and structure of the encoded proteins were analyzed.Transcriptome sequencing and qRT-PCR were used to analyze the expression characteristics of WRKY gene family members under drought stress.The results showed that 65 WRKY gene family members were identified and divided into three groups:Ⅰ,Ⅱ and Ⅲ,among which group Ⅱ was divided into five subgroups according to its structure and phylogenetic relationship.Analysis of WRKY gene structure and conserved motif showed that the number of exons ranged from 1 to 7,and the number of exons and motif distribution in the same subgroup were similar.The conserved domain of WRKY gene had certain variation,including the deletion of WRKY heptapeptide domain,the deletion of zinc finger structure,and the heptapeptide domain was mutated into WRKYGKK,WRKYGRK and WRKYGRK.Transcriptome sequencing data showed that 4 WRKY family members were not expressed in the samples,and 49 WRKY family members were up-regulated under drought stress,while 11 WRKY family members were down-regulated under drought stress.The above results showed that,chestnut WRKY genes played an important role in response to drought stress.

To provide a theoretical basis for the resource utilization of Chinese chestnut,the mature leaves of four chestnut cultivars were tested using ultra high performance liquid chromatography-tandem mass spectrometric,and functional secondary metabolites were screened by consulting relevant pharmacopoeias and searching the Chemical Book,Chemsrc,pharmacopeias and Web of Science database. The contents of 18 secondary metabolites we focused on in different cultivars were compared and analyzed. The study showed that a total of 673 kinds of secondary metabolites were detected in chestnut leaves,including flavonoids,phenolic acids,alkaloids,tannins,terpenoids,lignin and coumarin. Principal component analysis and cluster analysis showed that the difference of secondary metabolomics profile between Yanshanzaofeng and Yankui was little. The major secondary metabolites contributing to the classification were phloretin,didymin,salirepin,arbutin,cinnamic acid,gallocatechin,procyanidin B1 and so on. The differential metabolites could also be used as biomarkers to distinguish different chestnut varieties. A total of 202 functional secondary metabolites were selected,including 51 phenolic acids,24 alkaloids,83 flavonoids,21 terpenoids,14 lignins and coumarins,and 9 tannins. The contents of active substances were high in leaves such as protocatechuic acid,maslinic acid,ursolic acid,ellagic acid,chlorogenic acid,avicularin,kaempferol-7-O-glucoside,quercetin,spiraeoside,astragalin,betanin,tiliroside,ditymin and baimaside,which provided reference for the resource utilization of Chinese chestnut leaves.

To reveal the soil factors that formed the quality characteristics of Hebei chestnut,54 typical chestnut orchards from 8 counties in Hebei Province were selected as the objects,and the contents of 9 mineral elements in the soil were studied by using significance,correlation and principal component analysis.The results showed that there were no significant differences in contents of Mg and Na,but there were significant differences in N,P,K,Ca,Fe,Mn and Cu contents in the soil of chestnut plantations in the main chest nut production areas.The N content in the soil of chestnut orchards was generally below the level of 3,and the P content was generally above the level of 3.The K content was generally at a low level,which was below the level of 5.The correlation of the contents of metal elements such as Ca,Mg,Fe,Mn,Cu and Na were significantly correlated with each other in soil.They were also significantly correlated with P element,and the correlation with N content was generally weak.Principal component analysis showed that mineral elements in the first 3 principal components reflect the total information content 73.521%.The contribution rate of the first principal component accounted for 44.678%,and the elements which included Ca(0.871),Cu(0.840),Fe(0.809)and Mg(0.806)were the main factors.It showed that the metal trace element content contributes more to the principal component.The comprehensive score was based on the mineral elements content of different chestnut orchards,which decided the order of high and low.The highest score was in Qianxi County(0.591 8).The ranking of the soil quality in the main chest nut production areas was:Qianxi>Qinglong>Xinglong>Kuancheng>Shahe>Zunhua>Xindu>Funing.To sum up,it is confirmed that the comprehensive scores of metal elements contents such as Ca,Mg,Fe,Cu can be used to evaluate the soil quality by using principal component analysis.It was advisable to select high K and low P compound fertilizer,and it was recommended to apply multiple elements mixed fertilization as a supplementary measure to improve the soil quality level of chestnut orchards.

The suitable means to construct core collection of Yanshan chestnut germplasm based on SSR makers was studied, which was beneficial for preservation, management and utilization of chestnut germplasm resources.Taking 161 Yanshan chestnut germplasm resources from 10 cities(counties)as original materials, an allele-preferred sampling method and random sampling strategy were compared using UPGMA cluster method according to 3 genetic similarity coefficients(SM, Dice and Jaccard coefficient)based on SSR molecular markers. The effective number of alleles(Ne), Nei's gene diversity(H)and Shannon's information index(I)of two groups were compared to determine the optimum method. t -tests of core collection, initial collection and reserve collection were conducted to evaluate the representativeness of core collection. The principle coordinate analysis method and phonotypic traits analysis were used to confirm on the core collection.Compared with the random sampling strategy, allele preferred sampling method was more representative with higher values of genetic diversity indexes. The results showed the method of stepwise clustering according to SM similarity coefficient was better than Dice coefficient and Jaccard coefficient. 46 core collection included 28.57% initial collection. The effective number of alleles, Nei's gene diversity, Shannon's information index were respectively 1.531 7, 0.321 8 and 0.491 0. t -test showed the genetic diversity index of core collection was significantly higher that of initial collection.The core collection was evenly distributed in the principle coordinate diagram of initial collection, which could represent genetic diversity of the whole chestnut germplasm comprehensively.The method of allele preferred sampling and stepwise clustering according to SM similarity coefficient was a suitable means to construct core collection of Yanshan chestnut germplasm based on SSR makers, and 46 core collection could represent genetic diversity of original germplasm resources.

Genetic variations analysis of 127 Yanshan chestnut germplasms from 11 populations 15 phenotypic traits related catkin of Yanshan chestnut were designed with genetic diversity analysis,correlation analysis and principal component analysis,in order to provide valuable basic data and theory foundation for normalization,standardization,preservation and construction of chestnut core collections,provides the basis for the gene localization of agronomic traits. The results showed that:phenotypic traits related catkin of Yanshan chestnut were rich in phenotypic diversity,with mean variation coefficient (CV) of 27.55% and Shannon's diversity index(H')of 1.99. The mean variation coefficient(CV=64.80)of bisexual catkin numbers/catkin numbers was the largest,the mean variation coefficient (CV=7.52)of male catkin numbers/catkin numbers was the smallest. The Shannon's diversity index(H'=2.08)of stamen length was the highest,the Shannon's diversity index(H'=1.89) of bisexual catkin numbers/catkin numbers were the lowest. 9 phenotypic traits related to catkin were highly significant which indicated that a wide range of variation existed in these two levels. Correlation analysis showed that:More flower traits were highly significantly and extreme significantly related. Principal component analysis showed that floral traits in the first 5 principal components reflect the total information content 85.35%,bisexual catkin numbers/bearing shoot,catkin length/catkin width,catkin width,catkin length,bisexual catkin numbers/catkin numbers,male catkin numbers/catkin numbers,female flower numbers/bearing shoot,stamen length,floral axis length/floral axis width,9 characters was the main factor causing variation in traits of Yanshan chestnut. The 127 materials were divided into 8 kinds of cluster analysis. Flower character same source with similar material part can get together,the results were similar to principal component analysis.

To verify the diversity of fungal community structure in chestnut during storage, the fungal ITS amplitons of chestnut shell and pip from 6 species was sequenced by high-throughput method. It was found that the main pathogen of different chestnut variety differed greatly. Few were found in ancient chestnut in Mata village, Zhuyang town. Ancient chestnut in Tonghuagou village, Zhuyang town was Gibberella. Ancient chestnut in Dayu village, Huangqian town was Fusarium and Botryosphaeria. Ancient chestnut in Zhuanwayao, Xiagang town was Penicillium. Ancient chestnut in Honghe village, Jiaoyu town was Trichoderma. Castanea mollissima cv. Zaofeng was Penicillium and Colletotrichum. Community diversity index analyze showed that, fungus diversity, dominance, abundance and phylogenetic diversity of ancient chestnut in Mata village were the highest. Analysis within species found that, for ancient chestnut in Mata village, zhuyang town, the abundance of Fusarium in pip was significantly lower than that in shell. For ancient chestnut in Tonghuagou village, Zhuyang town, the abundance of Gibberella in pip was significantly lower than that in shell. For ancient chestnut in Honghe village, Jiaoyu town, the abundance of Penicillium in pip was significantly higher than that in shell. For C. mollissima cv. Zaofeng, the abundance of Penicillium in pip was significantly higher than that in shell, and the abundance of Colletotrichum was significantly lower than that in shell. At the same time, the fungal community structure of chestnut pip and shell was highly similar for ancient chestnut in Mata village, Zhuyang town, ancient chestnut in Dayu village, Huangqian town and C. mollissima cv. Zaofeng; and the fungal community structure of chestnut pip and shell differed greatly for ancient chestnut in Honghe village, Jiaoyu town and ancient chestnut in Zhuanwayao, Xiagang town.

The EST sequence of WRKY gene was obtained based on the transcriptome database of Castanea mollissima BL, and the cDNA(CmWRKY)sequence of WRKY transcription factor in chestnut was cloned by RT-PCR. The information of CmWRKY gene and its encoding protein were analyzed and the gene expression in the prokaryotic cell was carried out. The results showed that CmWRKY gene was 1 437 bp in length, encoding 479 amino acids with a calculated molecular weight of 52.128 96 ku and theoretical isoelectric point of 9.15. It had two WRKY conserved domains and two C2H2 zinc finger domains, belonging to Group Ⅰ of the WRKY family, and its gene accession number was KY312850.1. At the nucleotide level, the similarity of the CmWRKY gene with the corresponding sequence of Prunus persica was over 80%, and the highest similarity with the Qurcus suber WRKY gene was 97%. Homology modeling indicated that the tertiary structure of CmWRKY protein was similar to WRKY1 protein of Arabidopsis thaliana, suggesting that it might have the similar regulatory function as AtWRKY1 did. Molecular evolution analysis showed that chestnut CmWRKY was closely related to the WRKY of Qurcus suber and Juglans regia. SDS-PAGE analysis showed that the optimal expression condition of CmWRKY protein was 0.2 mmol/L IPTG induced at 30℃ for 10 h, and the molecular weight of the protein was about 56 ku, mainly in the form of soluble protein. The results laid the foundation for further studying the biological function and application of CmWRKY gene.

The experiment was to verify the effect of low temperature exotherms analysis on identifying the cold resistance of chestnut.The test materials were annual dormant branches of 16 chestnut varieties(strain) from 8 provinces,the exothermic characteristics of each varieties and the correlation index of LTE were analyzed by LTE analysis method.Low temperature exotherms(LTE) of 16 chestnut varieties(strain) were recorded by differential thermal analysis(DTA),the regression line of injury-temperature(LT-I) of xylem and phloem were established by three-point method,the LTE correlation index(LT-I slope of xylem and phloem,the temperature of freeze beginning and freeze ending point of xylem and phloem) and the membership function method were applied to compare cold hardiness of 16 chestnut varieties(strain).Tissue browning and conductivity method were used to validated the correctness of low temperature exotherms analysis method.The LT-I slope of phloem was different from xylem,the LT-I slope of xylem and phloem were different among 16 chestnut varieties,and the LTE correlation indexes had significant differences.There was highly significant negative correlation between the average membership of LTE index and the temperature of freeze beginning and freeze ending point of xylem and phloem.There was significant positive correlation between xylem injury degree determined by LTE analysis method and xylem browning rate determined by tissue browning,and the correlation coefficient was 0.988.There was highly significant positive correlation between branch injury degree determined by LTE analysis method and branch injury degree determined by conductometry,and the correlation coefficient was 0.998.The results show that LTE analysis is a simple,accurate and practical method for identifying the cold resistance of Chinese chestnut.The lower temperature of freeze beginning and freeze ending point of xylem and phloem,and the bigger average subordination value of LTE indexes in the chestnut branches is,the more cold resistance capacity the chestnut variety has.