Construction of core germplasm collection of Chinese chestnut cultivars (lines) based on SSR fluorescence markers

Abstract:【Objective】Chestnut is an important economic forest species, and there is a long history in chestnut cultivation and the nut is of high economic value. However, there are many cultivars (lines), and it is difficult to conserve the resources. Therefore, it is urgent to construct the core germplasm of chestnut cultivars (lines), so as to provide reference for innovative utilization of chestnut resources in the future.【Methods】With 342 Chinese chestnut cultivars as materials, 21 pairs of SSR fluorescent primers were used for amplification. The fragment position information in the mixtures was analyzed by capillary electrophoresis and the allele- specific fragment size information was read using Gene Marker v2.2.0 software (Soft Genetics LLC, State College, PA, USA). The genetic diversity and clustering analysis of the original chestnut cultivars (lines) germplasm were performed using the software of Powermarker. The phenotypes of clustering results were analyzed for significant differences using the software of Prism. In addition, based on the principle of maximum number of alleles, stratified sam-pling, simulated annealing algorithm and the random search algorithm were adopted to construct core collection. The difference significance analysis of genetic diversity indexes of core germplasm and original germplasm was performed using Prism software【Results】The results showed that 212 alleles were detected by 21 pairs of SSR primers, and the number of alleles (Na), effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) were 10.095, 3.488, 0.596, 0.658 and 0.642, respectively. The results showed that Chinese chestnut cultivars (lines) had rich genetic diversity. Cluster analysis showed that 342 chestnut cultivars were divided into two groups: the North and the South, and there were many hybrids, indicating that there were many gene exchanges between the South and the North. There were 42 resources in the mixed part, most of which were from Shandong province, with 33 resources accounting for 78.57% , which was also the merging area of two major groups in the North and South geographically. The water, soluble sugar, amylose, amylopectin and total starch contents of 19 phenotypic traits of chestnut cultivars (lines) in both south and north China were measured and the significant difference was analyzed. The results showed that there were significant differences in soluble sugar, amylose, amylopectin, prickly involucre thickness, nut shell thickness and nut width between the South and North cultivars (lines) of Chinese chestnut. There were extremely significant differences in total fruit weight, the weight, width, height and skin thickness of involucre and single nut weight. There was no significant difference among other indexes such as water and total starch contents, and kernel yield. Phenotypic data analysis showed that except for amylose content, kernel yield and nut shape index, phenotypes of other South cultivars (lines) were greater than those of North cultivars (lines), such as water, and soluble sugar contents, single nut weight and other phenotypes. According to the unique maturity period, branch length and curvature and spiny awn color of chestnut cultivars (lines), 8 cultivars (lines) with special traits were selected from all the resources as the required varieties, and these resources will play an important role in breeding and genetic research in the future. These eight resources are Jingshuhong, Yanshanzaofeng, Duanzhi No. 1, Duanzhi No. 3, Chaoduanzhi No. 2, Chuizhi No. 2, Wuhua and Shandonghongli. When the number of alleles was the largest, the number of samples for hierarchical sampling, simulated annealing algorithm, and random search algorithm was 342, 325 and 85, respectively. By comparing the number of samples calculated by the three methods, the simulated annealing algorithm based on the number of alleles was determined as the best sampling strategy, and finally 85 core collections were constructed. The principal coordinate analysis of the distribution of the core germplasm among all the germplasm resources was conducted, and the results showed that the core germplasm was distributed evenly in all the germplasm resources. And in PCA, principal coordinates 1 and 2 accounted for 34.36% and 15.71% of the variance in the locus information data. The analysis of genetic diversity parameters of core collection showed that the retention rates of core collection, total allele number (N), allele number (Na), effective allele number (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) were 24.85%,100%, 86.32%, 85.46%, 98.15%, 90.12% and 100.79%, respectively. Ttest results showed that there was no significant difference in genetic parameters between core and original collection, which could fully represent the genetic diversity of original collection.【Conclusion】 Based on SSR molecular markers, this study indicated that chestnut cultivars (lines) had rich genetic diversity, and revealed that there were more gene exchanges among chestnut cultivars (lines) between south and north China. In this study, the simulated annealing algorithm based on allele number was finally determined as the optimal sampling strategy, which was a good method for constructing chestnut cultivars and 85 core germplasm resources were obtained, laying a foundation for the effective conser-vation of chestnut cultivars resources. Almost all cultivars (lines) of Chinese chestnut were covered in this study. However, with the needs of the market and the development of science and technology, new excellent cultivars (lines) of Chinese chestnut resources will continue to appear. Therefore, the construction of core germplasm is a dynamic process, and it is necessary to continuously improve the core germplasm in future research.