Using Modified FIASCO Protocol to Isolate Polymorphic Microsatellite Loci in Chinese Brake Fern (Pteris vittata L.)：an Arsenic-Hyperaccumulating Plant
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摘要: 蜈蚣草（Pteris vittata L.）是目前用于砷污染土壤修复最好的超富集植物，但其分子水平上的研究数据较少。为了开发蜈蚣草特异性SSR遗传标记，本文采用改良的FIASCO方法从蜈蚣草AG和AC微卫星富集文库中随机挑选100个克隆，分离得到51个微卫星位点，其中60%为完美型（Perfect）SSR。根据这些位点设计、合成了25对引物，并对江西庐山及湖北恩施两地蜈蚣草种群各20个个体进行了遗传多样性检测，结果发现：其中8个完美型及1个间断型（intermittent）SSR位点的引物能够扩增出清晰、稳定且具有多态性的条带。9对引物共扩增出41个等位基因，各位点等位基因数在2~7之间，平均等位基因数为4.56个；期望杂合度在0.0494~0.8169之间；没有连锁不平衡现象发生。采用大叶井栏边草（Pteris multifida Poir.）进行跨种扩增，结果发现其中6对引物能够进行种间扩增。这些SSR分子标记的开发有助于蜈蚣草生态适应性进化分析、揭示蜈蚣草地理分布格局以及探讨蜈蚣草遗传多样性，还可用于品种鉴定及选育等。Abstract: Chinese brake fern (Pteris vittata L.) is the most important and well-known arsenic-hyperaccumulating plant used in phytoremediation of arsenic contaminated soils; however, little is known about its genetic diversity. In this study, 100 clones were randomly selected from the library enriched for AG and AC motifs using a modified FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) protocol and sequenced. Fifty-one microsatellite loci, of which 60% were pure repeats, were isolated. Twenty-five pairs of primers were designed and synthesized to evaluate their application and polymorphism in 20 individuals per sampling site obtained from Lushan and Enshi, respectively. Primers of eight loci of pure repeats and one locus of intermittent repeats were finally amplified successfully and yielded clear bands. A total of 41 alleles were detected. The allele number per locus of these microsatellites ranged from two to seven (mean 4.56). The expected heterozygosity (Exp-Het) ranged from 0.0494 to 0.8169. No linkage disequilibrium was found. Cross-species amplification demonstrated that six loci were amplified successfully in P. multifida. The markers helped to reveal the genetic variations of arsenic tolerant genotypes and understand the distribution pattern and ecological adaptation mechanism of P. vittata, and also assisted in breeding new varieties of fern for more effective remediation.
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