Abstract: 【Objective】The objective of this study to develop SSRs with high motif (tetra-, penta- and hexa-) nucleotide repeat and use them to evaluate the genetic diversity of common millet germplasms, and to provide significant implications for future linkage maps construction and evolution research of common millet.【Method】Comprehensive evaluation of phenotypic diversity was carried out based on membership function, principal component analysis (PCA) and cluster analysis. SSR primers developed in author’s laboratory by high-throughput sequencing were used to identify polymorphisms in different common millet accessions from various geographic origins. PowerMarker 3.25 and PopGen 1.32 were used to calculate the measures of genetic diversity and Nei’s genetic distance, respectively. MEGA 5.0 and Structure 2.2 were used to carry out cluster analysis and identify genetic groups.【Result】A set of 96 common millet accessions were used and seven phenotypic traits were investigated. Results showed that the genetic variation of tiller numbers and plant height were the most abundant. A total of 85 pairs of amplified primers (56.29%) showed high polymorphism among the 96 genotypes. A total of 71 tetra-nucleotide repeat SSRs were detected, accounted for 83.53%. Meanwhile, 10 (11.76%) penta- and 4 (4.7%) hexa- nucleotide repeat SSRs were identified, respectively. The size of 85 SSRs ranged from 100 to 450 bp with an average value of polymorphism information content (PIC) of 0.51. Resolving power (Rp) value varied from 1.00 to 5.75 (mean = 3.15). The average Rp value of tetra-, penta- and hexa- nucleotide repeat SSRs was 3.15, 2.8 and 4.0, respectively. Evaluating the distribution frequency of 85 SSRs based on Rp, it was observed that their ranges were 0-1, 1-2, 2-3, 3-4, 4-5 and 5-6, with 1 (1.18%), 15 (17.65%), 31 (36.47%), 20 (23.53%), 12 (14.12%) and 6 (7.06%) markers at each interval, respectively. Fifty-one markers accounting for 60% distributed at intervals of 2-3 and 3-4. A total of 232 alleles were amplified among 96 accessions by the 85 SSR markers. 2-3 alleles were generated by each locus, with an average of 2.7294 alleles. Sixty-two markers produced 3 alleles, and 23 markers produced 2 alleles. The range of gene diversity was 0.2842-1.0633, with an average of 0.7708. The range of PIC was 0.0400-0.7281, with an average of 0.4723. Genetic distance and genetic identity of common millet resources with different ecotopes were 0.0093-0.5052 (average=0.1798) and 0.6034-0.9907 (average=0.8485). Cluster analysis based on unweighted pair group method of mathematical averages (UPGMA) separated the 96 accessions into four groups (Northeast spring-sowing, Northern spring-sowing, Northern summer-sowing and Loess Plateau spring & summer-sowing ecotopes). A genetic structure assay indicated a close correlation between geographical region and genetic diversity. 【Conclusion】The present work developed a set of 85 tetra-, penta- and hexa- nucleotide repeat SSRs in common millet. These high motif microsatellite markers with high Rp value can distinguish different genotypes, generate high polymorphisms in polymerase chain reaction amplification. Based on the above molecular markers, the genetic diversity of Chinese common millet germplasms was assessed and those accessions from Northern spring-sowing and Loess Plateau spring & summer-sowing ecotopes are highly and genetically diverse.

Key words: common millet (Panicum miliaceum), high motif SSR, genetic diversity, clustering analysis, population structure