| Foxtail millet, Setaria italica Beauv., was also known as Italian, German, Chinese, Hungarian or Siberian millet, which was one of the most ancient cereal crops made great contribution to human civilization development and still grown in northern China, India and other developing countries as staple food. Foxtail millet was characterized with its drought tolerance, salt tolerance, infertile soil tolerance, highly efficiency of water utilization, relatively balanced necessary nutrition for human consummation and other special traits. But because of being a kind of regional-important crop, biotechnology genetic database of foxtail millet was almost vacant especially in the fields of specific molecular markers, and this hindered the genomic progress and advanced breeding of the crop.Microsatellites or simple sequence repeat (SSR), short stretches of tandemly, repeated 1 to 6 nucleotide sequences, were ubiquitously present in eukaryotic genomes. Microsatellites markers were co-dominantly inherited, highly polymorphic and conserved in correlative and interrelated species. It had been widely applied for studing of population structure, genetic linkage map, QTL location, assistant breeding selection. However, the actual use of microsatellites markers for analysis of foxtail millet had been very scarce, because of the lack of SSR primer sequences. Thus, there was an urgency to develop the suitable SSR markers from the genome of foxtail millet, so as to construct genetic linkage map and to analyze genetic diversity of Setaria species, which should lay a foundation for functional genomics of foxtail millet.The objectives of this work were to develop SSR markers of foxtail millet using libraries enriched respectivlely with microsatellites by biotinylated (AC)15, (AT)15, (AG)12, (TG)12 and (TC)12 oligoprobes cross-hybridization of Sau3AI-digested fragments of Yugu1 genomic DNA, with the assistance of affinity between biotinylated oligoprobes and Streptavidin Magnesphere Beads. Tatal of 2333 clones were obtained from the five libraries constructed. Then, all clones were secondly screened for SSR containning by double bacterial PCR with (AC)10, (AT)15, (AG)10, (TG)10 and (TC)10 oligoprobes as primer and their current vector primers. At last, 1106 clones from 1122 positive ones after screening were sequenced and 846 (76.5%, 846/1106) SSR containing sequences were obtained. After alignment comparison of sequences from the same library, 417 non-redundency sequences were identified which contained 463 SSR loci. Repeat type analysis of those microsatellites identified showed that the proportion of (AC/TG)n, (CA/GT)n, (AG/TC)n and (GA/CT)n (n≥5) repeats occurred relatively high, approximately 98.9% in all. There existed other motifs including dinucleotide repeats (AT/TA)n, tetranucleotides repeats (GACA)n, (ACGC)n, (GCGT)n and hexanucleotides repeats (ATACAC)n (n≥5). According to the principle of SSR primers designing, 393 SSR primer pairs were designed out of the 463 microsatellite containing non-redundency sequences. In addition, 25 primer pairs were designed by 16 YAC SSR-containing sequences from Georgia University, USA, and the total number of foxtail millet SSR primer pairs was 418.All the 418 SSR primer pairs were tested in Yugu1 for their amplification stability, and it revealed that most of the primer pairs could give stable amplification for the putative fragment in their first try, those that failed in their first try could also give proper amplification with optimized PCR reaction conditions. 39 SSRs with relatively fewer non-peculiar bands were selected to test their polymorphic behavior with 20 Setaria genotypes including 12 foxtail millet accessions and 8 green millet (S.viridis) accessions. 22 pairs of primers, out of the 39 tested, showed good amplification and polymorphism in the samples tested, and 185 alleles were identified totally, 8.41 per primer pair, with an average polymorphic information content (PIC) value of 0.76. Of the 22 polymorphic SSRs, primer pairs GTDZ7-73 and AGDZ23-41 identified the maximum16 alleles and AGDZ23-10 gave the minimum 2 alleles, whose PIC value were 0.91, 0.90 and 0.37, respectively. It explained that the SSR markers developed from genomic enriched libraries, which showed higher variation frequency, could be applied to genetic diversity analysis of Setaria species.To assay the utility of those markers developed, the chosen 22 primers were used to primarily study the genetic diversity of Setaria species. A total of 221 alleles were found after analyzing 28 individuals of 10 Setaria species, ranging from 2 to 22 with an average of 10.05 per primer pair. The mean PIC value of 0.78 based on the 28 genotypes, ranged from 0.37 to 0.93. Furthermore, SSR markers showed different variation in foxtail millet, green millet and other 8 Setaria wild species, whose total number of alleles was 138, 175 and 194 respectively, with the percentage of 62.4%, 79.2% and 87.8% in all of 221. While the mean PIC value of green millet (0.73) was a little higher than that in foxtail millet (0.72), which demonstrated the higher genetic variation of wild green millet accessions comparing with the cultivated foxtail millet accessions, and suggested the closer genetic relationship between them.The genetic similarities among accessions estimated using the SM coefficient and the phylogenetic evolution dendrograms based on the SSR data of this experiment were constructed using UPGMA (unweighed pair-group method with arithmetic averages) with the statistics software of NTSYSpc-2.11. As a result, it showed that all the 28 accessions were clearly distinguished and divided into two groups: Group Viridis and Group Glauca. The Viridis group included 12 S.italica, 8 S.viridis, 1 S. faberii, 1 S. verticillata, 1 S. leucopila and 1 S. queenslandica, and the Glauca group included 1 S. glauca, 1 S. parviflora, 1 S. pumila and 1 S. palmifolia. And that 12 S.italica accessions clustered together closely, showing the relatively nearer relationship and fewer genetic variation. Foxtail millet was shown closely related with green millet supporting the viewpoint that S.italica had originated from S.viridis. Whereas the 4 accessions falling into the Glauca group showed distant genetic background with the species fall into the Viridis group, which showed some evidence for the analysis of Setaria species evolution.This paper reported the first set of 418 pairs SSR primers development using microsatellite enriched libraries, and 22 primer pairs were analyzed in detail for their amplification and polymorphic behavior. Those primers designed were used in a primary study for genetic diversity and evolution analysis of Setaria species, so as to test their utility in related research. All the results demonstrated that those SSR primers could be used for genomic study of foxtail millet and its related species. |
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