| Foxtail millet (Setaria italica) is primarily grown in China as an important self- pollination crop for human consumption and also for forage. Although its diploid nature (2n=2x=18) and small genome size is suitable for genetic research, the genetic basis of most important traits of this crop is limited. Lacking of SSR markers is one of the reasons of its limited genetic background. Simple sequence repeat (SSR) markers are powerful tools in molecular studies; they have been widely used in genetic and marker-assisted breeding studies in rice, maize, wheat and most other important crops. The target of this research was designed to development SSR markers in foxtail millet.There many pathways for SSR development, but the 5′anchored PCR method is powerful for those crops of lacking genomic database, such as foxtail millet. Eight degenerate primers anchored by several basepairs in 5′, including four motifs [GT/CA, TG/AC, GA/CT, and AG/TC], were designed and used to amplify SSR regions of the foxtail millet cultivar Yugu1, and the amplification products were ligated to pGEM-T vectors, and eight genomic libraries enriched for microsatellites were constructed.As a result, a total of 229 clones were sequenced from the libraries constructed, and 140 were unique clones. The redundant rate was 31.0%. Most of those microsatellites contained short dinucleotide repeats with the average repeat number of 7.19, and the repeat number ranged from 6 to 31. The longest repeat was GA/CT motif with the average repeat number of 8.03, and the shortest was GT/CA with the average repeat number of 6.57.222 SSR primer pairs were designed against those SSR loci obtained, and 123 specific primer pairs sucessfully amplified putative fragment in Yugu 1. 14 primer pairs could amplify only in S.italica, and 109 primer pairs could successfully amplify across S.italica and S.viridis.Among those primer pairs designed, 56 polymorphic primer pairs were screened with S.italica and S.viridis. 6 primer pairs which amplified polymorphic bands in S.italica failed to amplify in S.viridis. 24 primer pairs which generated monomorphic patterns when tested in 8 S.italica materials amplified polymorphic bands in S.viridis samples. 26 primer pairs can give polymorphic patterns both in S.italica and S.viridis.22 single locus polymorphic SSR markers were used for diversity analysis among Setaria species. All markers could detect at least two alleles across the genus. The 22 SSR loci detected 95 alleles with an average number of 4.32 alleles per locus. The PIC value ranged from 0.093 to 0.76.The UPGMA dendrogram constructed with our 22 SSR markers clearly distinguished S. italica and other Setaria weed species. Compared to S. italica, Setaria weed species showed more allelic variation of microsatellite loci. Four species (S.viridis, S. italica, S.faberii and S.leucopila) showed close genetic relationship, but S.glauca and S.palmifolia showed lower genetic similarity with other species. These results demonstrated that SSR markers developed in this experiment can be used in most Setaria species.We successfully developed 123 SSR markers in foxtail millet using 5′anchored PCR method. 56 SSR markers could generate polymorphic patterns in 15 materials from S.italica and S.viridis. Those markers tested could transferablely use in other Setaria species. This is the first set of SSR markers developed in foxtail millet, which could be used for germplasm diversity and marker-assisted breeding studies among Setaria species.
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