Development of highly polymorphic simple sequence repeat markers using genome-wide microsatellite variant analysis in Foxtail millet [Setaria italica (L.) P. Beauv.]
- Shuo Zhang1,
- Chanjuan Tang1,
- Qiang Zhao2,
- Jing Li1, 4,
- Lifang Yang1,
- Lufeng Qie1, 4,
- Xingke Fan1, 4,
- Lin Li1, 4,
- Ning Zhang3,
- Meicheng Zhao1,
- Xiaotong Liu1,
- Yang Chai1,
- Xue Zhang1, 4,
- Hailong Wang1, 4,
- Yingtao Li1, 4,
- Wen Li1, 5,
- Hui Zhi1Email author,
- Guanqing Jia1Email author and
- Xianmin Diao1, 4Email author
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 3 September 2013
Accepted: 27 January 2014
Published: 28 January 2014
Foxtail millet (Setaria italica (L.) Beauv.) is an important gramineous grain-food and forage crop. It is grown worldwide for human and livestock consumption. Its small genome and diploid nature have led to foxtail millet fast becoming a novel model for investigating plant architecture, drought tolerance and C4 photosynthesis of grain and bioenergy crops. Therefore, cost-effective, reliable and highly polymorphic molecular markers covering the entire genome are required for diversity, mapping and functional genomics studies in this model species.
A total of 5,020 highly repetitive microsatellite motifs were isolated from the released genome of the genotype 'Yugu1’ by sequence scanning. Based on sequence comparison between S. italica and S. viridis, a set of 788 SSR primer pairs were designed. Of these primers, 733 produced reproducible amplicons and were polymorphic among 28 Setaria genotypes selected from diverse geographical locations. The number of alleles detected by these SSR markers ranged from 2 to 16, with an average polymorphism information content of 0.67. The result obtained by neighbor-joining cluster analysis of 28 Setaria genotypes, based on Nei’s genetic distance of the SSR data, showed that these SSR markers are highly polymorphic and effective.
A large set of highly polymorphic SSR markers were successfully and efficiently developed based on genomic sequence comparison between different genotypes of the genus Setaria. The large number of new SSR markers and their placement on the physical map represent a valuable resource for studying diversity, constructing genetic maps, functional gene mapping, QTL exploration and molecular breeding in foxtail millet and its closely related species.
KeywordsMicrosatellite marker SSR development Polymorphism Setaria italica
Foxtail millet (Setaria italica) is an ancient crop that is grown worldwide in arid regions, especially in East and South Asia, Africa and Europe [1–3]. According to data from the Food and Agriculture Organization, about 30 million tons (Mt) of millet grain are produced annually (http://faostat.fao.org/). In China, the current annual growing area of foxtail millet is over 2 million hectares, with a grain yield of 6 Mt . As a drought-tolerant crop, foxtail millet has the potential to become more important, especially as the climate is becoming warmer and dryer [4–6].
Its small diploid genome (~515 Mb) and inbreeding nature has led to foxtail millet becoming a model for grass functional genomics, especially in investigating plant architecture, drought tolerance, crop domestication, C4 photosynthesis and the physiology of bioenergy crops [7–9]. The release of the genome sequence [10, 11] and a haplotype map  have made the use of foxtail millet as a model species more attractive.
Simple sequence repeats (SSRs), also known as microsatellites, are tandem repeats of 1 to 6 nucleotides that are present in both coding and non-coding regions [13, 14]. SSRs have become a marker of choice in genotyping because of their high abundance, high level of allelic variation, co-dominant inheritance and analytical simplicity. Moreover, microsatellite markers could be effectively applied in phylogenetically related species according to their conserved sequences among diverse organisms, which will greatly benefit genetic studies of related species . However, despite the use of both genomic [16–19] and transcriptional  sequences for generating SSRs, the number of SSR markers in foxtail millet is still not adequate for efficient genetic analyses and gene mapping studies.
The level of polymorphism of SSRs is a key factor for their efficient application, and can be affected by a number of factors, including the nucleotide motif and repeat number. SSR polymorphisms are positively correlated with the number of repeat units . As reported in humans , rice [23, 24] and Medicago truncatula Gaertn , SSRs with higher numbers of repeats tend to be more polymorphic.
The availability of the completed genome sequence of foxtail millet [10, 11] provides an ideal resource for genome-wide identification of SSRs in silico and the development of locus-specific SSR markers in this species. Taking advantage of this resource, we identified a large number of highly polymorphic SSRs by scanning for microsatellite units with relatively higher repeat numbers in the foxtail millet genome, and then assessed the efficiency of their application as developed SSR markers. The polymorphism information content (PIC) values of the SSR markers were also characterized by amplifying genotypes of a set of Setaria accessions from diverse geographic origins. These SSR markers could significantly stimulate genetic and genomic studies of foxtail millet and related species, further promoting it as a novel model system for genomic study.
Identification of microsatellite motifs in the foxtail millet genome and polymorphic SSRs determination
Number of polymorphic SSRs among 'Yugu1’, 'Daqingjie’ (DQJ) and 'N10’, and designed primers
Number of SSR sequences
Polymorphic vs. DQJ
Polymorphic vs. N10
SSR primer design
Number of Polymorphic SSRs
Percentage of polymorphisms
Based on the polymorphic microsatellite sequences identified above, 788 pairs of SSR primers were designed. Their distributions among the different chromosomes were different. The largest number was located on chromosome 9 (133), followed by chromosomes 6 (123) and 7 (94). Although there were many microsatellites in chromosome 8, only 35 pairs of SSR primers was designed because of the fewer genomic variants detected in chromosome 8 compared with the other chromosomes (Table 1).
Validation of application efficiency and transferability of SSRs among related species of foxtail millet
Construction of a physical map of the novel SSR markers
A foxtail millet SSRs database enriched with 733 pairs of novel polymorphic SSR markers
SSRs have become a powerful marker system for genotype analysis, diversity estimation, QTL mapping and other related genetic and genomic studies . However, the number of highly polymorphic SSR markers developed for foxtail millet was limited. The first set of 26 expressed sequence tag (EST)-SSRs in foxtail millet was developed in 2007 , which was followed by four sets of genomic SSR studies that developed 190, 45, 170, and 21,294 SSRs, respectively, using microsatellites enriched libraries [16–18] and released reference genome sequences . Thus, a large set of SSRs was available for foxtail millet, which had the potential to meet the requirement of constructing high-resolution genetic maps for this model crop. However, only a small set of about 160 SSRs were evaluated for their PIC values in the Setaria genus in those works [16–20]. In the present study, 788 pairs SSRs were developed and all those markers were characterized based on 28 Setaria samples for their amplification efficiencies and polymorphism contents. Among them, 733 showed stable amplification and were highly polymorphic, with clear and available PIC values, allowing them to be anchored in the foxtail millet physical map. This large set of highly polymorphic SSR markers, combined with their corresponding physical locations, represent a valuable resource for genetic linkage map construction, QTL exploration, map based gene cloning and marker-assisted trait selection in this species. Furthermore, genome variant analysis could also be applied in studies of development of practical markers in other crop species.
Polymorphic performance of the newly developed SSR markers
According to the polymorphism evaluation of SSRs in rice  and maize , dinucleotide repeat unit microsatellites always have larger repeat numbers and show high level of polymorphisms. Correspondingly, the dinucleotide type of SSRs developed in this study had a high average PIC value of 0.68, which was the same as that reported by Jia . These values are significantly higher than those reported by others in foxtail millet [17, 18, 20]. This higher polymorphism performance implied that these markers could be used efficiently in foxtail millet genetic studies.
The frequency polymorphisms in GC & CG dinucleotide repeats detected in this study were low (Additional file 2: Figure S1), and similar to those reported in other crops [24, 29, 30]. This might be because GC-rich regions are relatively stable, resulting in less replication slippage, which generates the repeated motifs of SSRs , or because GC motifs are usually distributed in exons, where polymorphisms occur less frequently .
The majority of the highly polymorphic SSRs identified in this study were distributed in the non-coding regions of the foxtail millet genome (Additional file 5: Figure S3A, S3B). This might be a specific characteristic of highly polymorphic markers. Surprisingly, a larger proportion of the 'Tri’ type of SSRs was identified in coding regions, implying that three nucleotide insertions/deletions might be more acceptable for organisms to maintain regular growth under pressure from genomic variants occurring in coding regions. However, this hypothesis needs to be verified.
Transferability of the developed SSRs to related species
Most of the SSR markers developed from the genome sequence of the foxtail millet cultivar 'Yugu1’ could be used in green foxtail. As the latter is the wild ancestor of domesticated foxtail millet , the transferability of the SSRs indicates that they share a very similar genome, although they are classified as different species botanically . The phylogenetic analysis of the diverse Setaria accessions identified three gene pools, implying that the wild ancestor, domesticated landraces and improved cultivars of S. italica are distinct gene resources for breeding programs of foxtail millet. This observation is similar to that made in rice  and maize . Previous studies of the molecular diversity of Chinese foxtail millet  and green foxtail  also support this conjecture. A large proportion of the SSRs developed in this study could also be used in S. faberii and S. verticillata, probably because these two species share the AA genome with foxtail millet. Only 44.7% of the SSRs developed in this trial could be used in S. adhaerans and S. glauca, indicating their genetic distinction from the foxtail millet AA genome. These results were consistent with those from genomic in situ hybridization analysis of the Setaria genomes . Thus, the SSR markers developed in this study could be efficiently used in other closely related Setaria species.
This work represents a major advance in the identification and confirmation of SSR markers for Setaria. A large set of 733 highly polymorphic SSR loci, with an average PIC value of 0.67, were identified by genome variants analysis based on second-generation resequencing technology.
The reference genome sequence of the foxtail millet genotype 'Yugu1’ was retrieved from phytozome (http://www.phytozome.net/). SSRHunter  and MicroSAtellite (MISA) were used to identify microsatellite motifs (http://pgrc.ipk-gatersleben.de/misa), with the following search criteria: twenty repeat units for mononucleotide (Mono) repeats, eight (five for chromosome 6) for dinucleotide (Di) repeats, eight for trinucleotide (Tri) repeats and tetranucleotide (Tetra) repeats, and six for pentanucleotide repeats (Penta) and hexanucleotide repeats (Hexa). All selected microsatellites containing fragments were validated using the BLASTN tool in the software package ncbi-blast-2.2.25 + -win32.exe (downloaded from http://www.ncbi.nlm.nih.gov/guide/). According to the scores of all alignments for each query, a single copy was defined as the query with a top score significantly higher (at least five fold higher) than the second one. Only single copy sequences were selected for further analysis.
Selection of polymorphic SSRs
The S. italica accession 'Daqingjie’ (DQJ) and the S. viridis accession 'N10’ were resequenced using second-generation sequencing technology with high level coverage, and the sequences obtained were de novo assembled . The diffseq program (with default parameters) in the EMBOSS package  was used to compare sequence variants between the two de novo assemblies against the SSR sequences identified from the reference genome of 'Yugu1’. MUMmer3.22 (http://mummer.sourceforge.net/) was used to align all SSR-containing sequences with assemblies of 'DQJ’ and 'N10’, respectively, and a Perl Script was used to list the length polymorphisms. SSR containing sequences that showed polymorphisms among these genotypes were selected for primer design. Those primers that amplified a fragment between 100 bp and 300 bp were selected for further validation. Primer 3.0 (http://frodo.wi.mit.edu/) was used to design primers flanking the sequences of each unique SSR.
Amplification efficiency and polymorphism characterization
Sampled accessions for SSRs characterization in Setaria
Accession no. or cultivar
Foxtail millet, landraces
Inner Mongolia, China
Foxtail millet, cultivars
Inner Mongolia, China
Other setaria species
Physical map construction
BLASTN online (http://www.phytozome.net/search.php) was used to determine the physical position of each of the polymorphic SSR primers on the 'Yugu1’ genome, and the physical distances between adjacent SSRs were calculated manually. MapDraw  was used to construct a physical map including all the developed polymorphic SSRs.
Simple sequence repeat
Quantitative trait locus
Polymorphism information content
Expressed sequence tag.
The authors thank Dr. Chunji Liu from the Commonwealth Scientific and Industrial Research Organization, Canberra, Australia, for helpful discussions and for modifying the English in the manuscript. This work was supported by the National High Technology Research and Development Program of China (863 Program) (2013AA102603), the National Natural Science Foundation of China (31171560, 30630045, 31301328), Fundamental Research Funds of ICS-CAAS (Grant to Guanqing Jia, 2013007), and the China Agricultural Research System (CARS07-12.5-A02).
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