柑橘全爪螨微卫星位点鉴定与信息分析

doi:10.3864/j.issn.0578-1752.2016.02.009

摘要/Abstract

摘要： 【目的】通过磁珠富集法构建柑橘全爪螨（Panonychus citri）微卫星富集文库，发掘柑橘全爪螨基因组微卫星（gSSR）序列。同时，利用柑橘全爪螨转录组数据库，筛选其功能微卫星（EST-SSR）分子标记，设计柑橘全爪螨微卫星（SSR）引物并验证其适用性。【方法】在提取柑橘全爪螨高质量基因组DNA的基础上，使用链霉亲和素偶联的磁珠与生物素标记的微卫星探针结合，亲和性捕捉由限制性内切酶酶切产生的含SSR的单链基因组DNA目的序列，经PCR扩增为双链后，克隆并构建SSR富集文库，测序筛选SSR序列。另一方面，基于柑橘全爪螨转录组数据，利用msatcommander软件发掘其功能微卫星标记。采用Primer Premier 5软件设计SSR引物，并进行验证。【结果】构建了柑橘全爪螨AC、TC和ATG共3个SSR富集文库，阳性克隆率分别约为30%、28%和25%。对文库的测序结果表明，AC文库的冗余率最高，TC文库次之，而ATG文库未发现相同微卫星位点的克隆。同时，在AC文库中，大部分AC重复类型的SSR为多拷贝微卫星位点，其中有相当一部分SSR的一侧侧翼序列完全相同或高度相似。从3个SSR富集文库中获得了可用于设计引物的gSSR单一序列44条（GenBank登录号为JF776418—JF776461），共合成了20对SSR引物，其中能够稳定扩增的引物有11对。柑橘全爪螨gSSR中完美型SSR占54.5%，非完美型和复合型SSR分别占27.3%和18.2%。在完美型的gSSR中，两碱基重复SSR的核心重复次数（13—42次）大于三碱基重复SSR（5—9次）。从柑橘全爪螨转录组数据库中共获得8 023个EST-SSR位点，其中2 540个SSR可用于引物设计，共合成了35对引物（GenBank登录号为KT261306—KT261340），其中能够稳定扩增的引物有8对。柑橘全爪螨EST-SSR的平均分布频率为1/3.55 kb。其中，三碱基为优势核心重复类型，占总数的53.86%；其次为两碱基核心重复类型，占总数的43.36%；而四、五、六碱基重复类型以及复合型的SSR数量均较少，且数量差异不大，共占EST-SSR总数的2.78%。柑橘全爪螨EST-SSR核心重复次数主要集中在5—10次。【结论】采用磁珠富集法，并将酶切、接头连接一步化，可提高个体微小的螨类及微小昆虫SSR的富集效率。柑橘全爪螨gSSR核心重复次数要远多于从转录组数据库获得的EST-SSR。总体而言，gSSR和EST-SSR中的三碱基重复SSR具有更好的优化率。此外，柑橘全爪螨gSSR具有微卫星家族现象。

关键词: 柑橘全爪螨, 叶螨科, 高通量测序, 分子标记, 微卫星富集文库

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

魏丹丹，刘燕，杜洋，李刚，李婷，袁明龙，王进军. 柑橘全爪螨微卫星位点鉴定与信息分析[J]. 中国农业科学, 2016, 49(2): 282-293.

WEI Dan-dan, LIU Yan, DU Yang, LI Gang, LI Ting, YUAN Ming-long, WANG Jin-jun. Analysis of Microsatellite Loci from Panonychus citri Based on Enriched Microsatellite Library and Transcriptome Dataset[J]. Scientia Agricultura Sinica, 2016, 49(2): 282-293.

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