Abstract: 【Objective】 The objective of this study is to construct the microsatellite-enriched libraries, and identify genomic- microsatellite (gSSR) from the genome of the citrus red mite, Panonychus citri. Meanwhile, a large number of gene-microsatellite (EST-SSR) markers were also indentified from the transcriptome database of P. citri. Based on these SSR sequences, the selected SSR primer pairs were validated.【Method】On the basis of isolating high quality genomic DNA of P. citri, enriched microsatellite libraries were constructed by streptavidin-coated magnetic beads utilizing methodologies that exploit the strong affinity between biotin and the protein streptavidin. A fast and easy protocol was proposed through a combination of two different published methods. Briefly, genomic DNA was digested by the restriction enzyme and then ligated to designed adaptors. Microsatellite-containing DNA fragments were captured by streptavidin-coated magnetic beads. The beads affinity capture of microsatellite repeats using biotinylated oligonucleotide probes. Subsequently, PCR was used to amplify the captured molecules for transferring single strand DNA to double strand DNA. The PCR products were then ligated to pGEM-T Easy vector and transformed into Trans5α competent cells. Then, clones from these libraries were screened for microsatellite content. Meanwhile, msatcommander software and bioinformatics methods were used to identify and assess of the quality and quantity of EST-SSR loci from P. citri transcriptome dataset. Primer Premier 5 was used to design P. citri SSR primers, and then these primer pairs were verified by PCR.【Result】Three microsatellite-enriched libraries of AC-repeat, TC-repeat, and ATG-repeat were constructed for P. citri. The positive clone rates of these three libraries were about 30%, 28% and 25%, respectively. The sequencing results showed that the AC library had the highest redundancy rate, and the TC library followed. However, the same clone of SSR in the ATG library was not found. Intriguingly, in the AC library, some AC-repeat types of SSRs existed in many copies with similar or almost identical sequences in one of the flanking regions. Totally, 44 unique microsatellite loci (GenBank number JF776418-JF776461) were obtained. Among these SSRs, 20 primer pairs were synthesized, and 11 primer pairs could be steadily amplified. In gSSR, perfect SSR accounted for 54.5%, and imperfect perfected and compound SSR accounted for 27.3% and 18.2%, respectively. In perfect gSSR, the repeat times (13-42 times) of the di-nucleotide repeat SSR were much higher than tri-nucleotide repeat SSR (5-9 times). A total of 8 023 EST-SSR loci were identified from P. citri transcriptome, and 2 540 SSR sequences could be used for primer design. A total of 35 primer pairs were synthesized (GenBank number KT261306-KT261340), and 8 primer pairs could be steadily amplified. The average distribution distance of the transcriptomic SSRs was 3.55 kb. Tri-nucleotide repeat SSR was the most frequently occurring type in P. citri EST-SSR (53.86%), and di-nucleotide repeat SSR was followed (43.36%). The tetra-, penta-, hexa- nucleotide repeat and compound SSR were very scarce with similar numbers, and accounted for 2.78% in total. The repeat of times of the EST-SSR motifs were mainly concentrated in 5-10 times.【Conclusion】Microsatellite enrichment by magnetic beads is a suitable method to obtain genomic-microsatellite for the P. citri. DNA digestion and ligation were performed simultaneously, which can enhance the concentration of enzyme-digested products and the efficiency of microsatellite enrichment for the mite or small size insects. In P. citri, the repeat times of the gSSR were much higher than that in transcriptomic SSR. In general, the tri-nucleotide repeat SSR will be more suitable for the study of population genetic structure of P. citri. In addition, gSSR have microsatellite DNA families, which indicated that the microsatellite sequences exist in multiple copies in the genome of P. citri.

Key words: Panonychus citri, Tetranychidae, high-throughput sequencing, molecular marker, enriched microsatellite library