Development and characterization of genome-wide microsatellite molecular markers for Chinese chestnut

Chinese chestnut is an important economic forest tree species with enormous application value in the wood, medical, and chemical industries. Currently, the limited genome-wide SSR molecular marker information on chestnut resources significantly restricts research on the genetic diversity and identification of chestnut resources. To address this issue, we used GMATA to screen simple sequence repeat (SSR) markers throughout the Chinese chestnut genome. A total of 312,302 molecular markers were obtained with a density of 434.38 Mb^–1. Subsequently, all SSR markers were examined for polymorphism using the HipSTR program and 138,208 polymorphic loci were ultimately obtained. To verify the capability of the developed SSR for identification, we randomly selected 36 markers on 12 chromosomes to construct fingerprint maps of 96 ancient Chinese chestnut resources from the Yanshan Mountains. The results showed that only six pairs of primers were required to create a unique DNA fingerprint of the tested ancient trees, showing that the developed markers have good potential for identification. We then evaluated the inter-specific universality and polymorphism of these markers using 91 Castanea plants of three different species. The molecular markers amplified 94% of the interspecies with a polymorphic information content (PIC) value of 0.859. Cluster analysis revealed that the resources can be well differentiated using these developed markers, and these markers can be widely used to identify interspecific boundaries. The results of this study proved that the developed molecular markers have the potential for assessing genotypic diversity, which can provide references for genetic diversity research, variety identification, kinship analysis, the selection of good products, and the construction of core germplasm resources of chestnut and even chestnut plants. Also these markers provide a solid foundation for the molecular design of hybrids, improved breeding and development of germplasm resources.