Genome Survey and Large-scale Isolation of Microsatellites through Illumina Sequencing from the Red Swamp Crayfish Procambarus clarkii
Genome Survey and Large-scale Isolation of Microsatellites through Illumina Sequencing from the Red Swamp Crayfish Procambarus clarkii
Xiao Zhang1, Guoqing Shen1, Yang Wang1, Pengdan Huang1,2, Yan Shui2, Zenghong Xu2 and Huaishun Shen1, 2*
ABSTRACT
Microsatellites, also known as simple sequence repeats (SSRs), are a type of DNA marker that are highly polymorphic in the genome. However, the microsatellite markers available for the red swamp crayfish Procambarus clarkii are limited. In this study, a sizeable number of microsatellites were identified using the data of the P. clarkii genome survey generated from Illumina Hi-Seq PE sequencing. A total length of 1,625 Mb genomic sequences of P. clarkii were assembled based on 124 Gb raw sequencing data. Based on a Kmer analysis, the P. clarkii genome size was projected as approximately 3.6 Gb. A total of 336,275 SSRs (simple sequence repeats) identified motifs were identified. There were 167,999 the most common type di-nucleotide repeat motifs (49.94%). The second most common repeats were the tri-nucleotide ones (131,405, 39.09%). A total of 312,454 microsatellite loci had flanking sequences. A subset of 56 primer pairs was randomly selected for the PCR experiments to validate the SSRs that had been identified. Forty-six primer pairs (80%) had PCR products that matched the sizes expected, and the products from 40 primer pairs (71%) exhibited polymorphism. Thirty-nine P. clarkii individuals were analyzed for their primer sets, and 22 were identified. The average allelic number in the 22 polymorphic loci was 3.6. The 22 microsatellite loci all had good polymorphisms. Among them, the polymorphism information content of the 12 loci is greater than 0.5, which is highly polymorphic.
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