Summary of all 77,996 microsatellite markers..xlsx
Note: All these 77,996 microsatellite markerswere developed based on the sequence of Gossypium hirsutum L. acc. TM-1 (Zhang et al. Nature Biotechnology 2015, 33: 531-537). The detail procedures of developing these microsatellite markers are reported in Scientific Reports (Wang et al. 2015, 5:10638 http://www.nature.com/srep/2015/150601/srep10638/full/srep10638.html ).
Identification of microsatellites
Genome sequences were searched for perfect microsatellites using PERL5 script MIcroSAtellite (MISA, http://pgrc.ipk-gatersleben.de/misa/) with basic motifs from mono- to hexanucleotide. Repeats with a minimum of 18, 9, 6, 5, 4, and 3 were defined for the mono- to hexanucleotide, respectively. Compound microsatellites were defined as ≥ 2 repeats interrupted by ≤ 100 bp.
Design of SSR primers
Primer pairs were designed from the flanking sequences of identified microsatellites using PRIMER3 software (Untergasser, A. et al. Nucleic Acids Res. 2012, 40:e115), and two perl scripts, p3_in.pl and p3_out.pl served as interface modules between MISA and Primer3 with the primer designing parameters: 18–27 bp in length, 57–63℃ in melting temperature, 30–70% in GC content and 100–280 bp in product size. These two perl scripts were downloaded from MISA(http://pgrc.ipk-gatersleben.de/misa/). Primer3 was downloaded from http://www-genome.wi.mit.edu/genome_software/other/primer3.html. The p3_in.pl was used to create a primer3 input file which was submitted to Primer3. Then p3_out.pl was used to calculated and merge all information together.