The Introduction of Hi-SSRseq

Microsatellites (short tandem repeats, STR, or simple sequence repeats, SSR) are widely used markers in population genetics. Despite accurate and efficient genotyping of SSRs constitutes the basis of SSRs as an effective genetic marker with various applications, the lack of a high throughput technology for SSR genotyping has limited their use as genetic targets in many crops. single‐nucleotide polymorphisms (SNPs) or insertions/deletions (indel) polymorphisms in the nucleotide sequence of that fragment, either within the repetitive array or in the flanking regions (FR), remain undetected by length assessment alone. Moreover, indels in the flanking regions might be incorrectly confounded with size mutations of the SSR.

As a consequence, the traditional assessment of fragment length may lead to underestimating genetic variability, inaccurate results, or even wrong evolutionary interpretations. To overcome such errors, information about the nucleotide sequence of each allele is needed. CD Genomics provided a technology called Hi-SSRseq that combined the multiplexed amplification of traditional SSRs with high throughput sequencing. This method can genotype plenty of SSR loci in hundreds of samples with highly accurate results, due to the substantial coverage afforded by high throughput sequencing, which also greatly reduces the cost and time of genotyping, and the comparison between samples can be directly based on the base sequence.

Our objectives were (a) to generate nucleotide sequence data of several non‐model plant species, for which prior genomic data did not exist, from both the SSR and the flanking regions, (b) to record the length of the repetitive region, as well as SNP and indel variation within the SSR and the FR, (3) to estimate the amount of molecularly accessible size homoplasy of each locus, and (4) to compare the degree of genetic variability between different datasets based on the number of repeat units, fragment length, and sequence identity.


  • Genetic analysis
  • Fine mapping
  • Quantitative trait locus (QTL) mapping
  • Marker-assisted selection (MAS) breeding

Key Features and Advantages

  • High Throughput: plenty of SSR loci can be genotyped in hundreds of samples with highly accurate results.
  • Cost Effective: Assay costs significantly less than most of other SSR genotyping platforms.
  • Simplified Hand-on Workflow.
  • More Accurate Results: Avoiding underestimating genetic variability, wrong evolutionary interpretations.

Project Workflow

Project Workflow

Service Specifications

Sample Requirements
  • DNA amount ≥ 50 ng(Single panel)
  • DNA concentration ≥ 20 ng/μl
  • Sequencing coverage: 1000X~5000X (According to the chromosome multiple of the species, such as diploid 1000 X; tetraploid 2500 X; hexaploid 5000 X)
  • Illumina MiSeq platform (2 × 250 bp paired‐end)
Data Delivery
  • The original sequencing data
  • Experimental results
  • Data analysis report

With state-of-the-art sequencing platforms and deep collaboration with highly experienced technicians and scientists across departments in CD Genomics, an Hi-SSRseq technique is offered that allows to genotype hundreds of individuals at many custom‐designed SSR loci simultaneously, combining multiplex PCR and Illumina sequencing. If you have additional requirements or questions, please feel free to contact us.


  • Petra Šarhanová, Simon Pfanzelt, Ronny Brandt, Axel Himmelbach and Frank R. Blattner. SSR-seq: Genotyping of microsatellites using next-generation sequencing reveals higher level of polymorphism as compared to traditional fragment size scoring. Ecology and Evolution. 2018;8:10817–10833.
  • Jingjing Yang, Jian Zhang, Ruixi Han, Feng Zhang, Aijun Mao, Jiang Luo, Bobo Dong, Hui Liu, Hao Tang, Jianan Zhang and Changlong Wen. Target SSR-Seq: A Novel SSR Genotyping Technology Associate With Perfect SSRs in Genetic Analysis of Cucumber Varieties. Frontiers in Plant Science. 2019; 10:1-12.
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