Identification of massive molecular markers in Echinochloa phyllopogon using a restriction-site associated DNA approach

Echinochloa phyllopogon proliferation seriously threatens rice production worldwide. We combined a restriction-site associated DNA(RAD) approach with Illumina DNA sequencing for rapid and mass discovery of simple sequence repeat(SSR) and single nucleotide polymorphism(SNP) markers for E. phyllopogon. RAD tags were generated from the genomic DNA of two E. phyllopogon plants, and sequenced to produce 5197.7 Mb and 5242.9 Mb high quality sequences, respectively. The GC content of E. phyllopogon was 45.8%, which is high for monocots. In total, 4710 putative SSRs were identified in 4132 contigs, which permitted the design of PCR primers for E. phyllopogon. Most repeat motifs among the SSRs identified were dinucleotide(>82%), and most of these SSRs were four motif-repeats(>75%). The most frequent motif was AT, accounting for 36.3%-37.2%, followed by AG and AC. In total, 78 putative polymorphic SSR loci were found. A total of 49,179 SNPs were discovered between the two samples of E. phyllopogon, 67.1% of which were transversions and 32.9% were transitions. We used eight SSRs to study the genetic diversity of four E. phyllopogon populations collected from rice fields in China and all eight loci tested were polymorphic.