To understand the genetic relationships of Castanea species, 16 phenotypic traits were measured, simple sequence repeat (SSR) markers were analyzed, and molecular identity cards (IDs) were constructed for 118 Castanea materials using fluorescent capillary electrophoresis. The coefficient of variation values of the 16 morphological traits of the test materials ranged from 11.11% to 60.38%. A total of 58 alleles were detected using six pairs of SSR core primers, with an average number of 9.7 alleles per locus. The average number of valid alleles per locus was 3.9419 and the proportion of valid alleles was 40.78%. A total of 105 genotypes were detected, and the number of genotypic species that could be amplified per primer pair ranged from 8 to 26. The mean value of the observed heterozygosity was 0.4986. The variation in the He, H, and PIC values was similar; the size of I value was approximately 2.21 times larger, and its mean number of variations was 0.7390, 0.7359, 0.6985, and 1.6015, respectively. The classification of 118 Castanea species was performed using three analytical methods: structure analysis, neighbor-joining (NJ) cluster analysis, and principal coordinate analysis (PCoA), and the results of the three methods were in high agreement. Six pairs of SSR core primers with high polymorphism and strong discriminatory properties were used to identify 118 Castanea plants, and a unique molecular ID card was constructed for each material. These results provide insight into the genetic diversity and population structure of Castanea plants and a theoretical basis for improving the phenomenon of mixed varieties and substandard plants in the Castanea plant market.

The chestnut is widely cultivated fruit tree enriched with nutrients and has pleasant tasting fruit. The starch properties of kernels greatly influence the nutritional quality of chestnuts. Although various studies have examined the production and accumulation of starch in chestnuts, the transcription alteration linked with starch properties in the kernels has not yet been fully assessed. The present study was conducted to compare transcriptomic variation and starch profiling of three chestnut cultivars: “Garrone rosso” (eur), Castanea sativa Mill., native to Europe; “Dahongpao” (dhp) and “Jiandingyouli” (jdy), and Castanea mollissima Bl., native to China. The results revealed that “dhp” and “jdy” had higher amylose, amylopectin and total starch content than “eur”. Based on transcriptome data, we screened 63.17G clean bases, and detected numerous differentially expressed genes (DEGs) that were associated with starch and sucrose metabolism. Through the combined transcriptomic and starch profiling analysis showed that DEGs in “the starch and sucrose metabolism”, “bZIP transcriptional factors”, and “zinc finger protein” pathways were positively correlated with starch accumulation, genes encoding sucrose synthase (CMHBY215664 and CMHBY203813), USPase (CMHBY206855), and PGI (CMHBY200699) were found to participate in the biosynthesis, transport, and regulation of starch according to their expression patterns in chestnut kernels. Furthermore, genes encoding different transcription factors (ERF, bZIP, MYB, and WRKY) that potentially regulate the expression of genes involved in starch and sucrose metabolism were selected by coexpression analysis, which highlighted that most MYB and WRKY TF members were positively correlated with starch synthase (SS). qRT-PCR assay results of nine selected DEGs confirmed the accuracy of the RNA-Seq data. Our results provide insights into genetic resources for deciphering the molecular mechanisms of chestnut starch accumulation.