Li Lun, Fang Zhiwei, Zhou Junfei, Chen Hong, Hu Zhangfeng, Gao Lifen, Chen Lihong, Ren Sheng, Ma Hongyu, Lu Long, Zhang Weixiong, Peng Hai
Institute for Systems Biology, Jianghan University, Wuhan, Hubei 430056, China.
Center for Development of Science and Technology, Ministry of Agriculture, P.R. China, Beijing 100122, China.
Nucleic Acids Res. 2017 Jun 2;45(10):e88. doi: 10.1093/nar/gkx093.
Accurate and efficient genotyping of simple sequence repeats (SSRs) constitutes the basis of SSRs as an effective genetic marker with various applications. However, the existing methods for SSR genotyping suffer from low sensitivity, low accuracy, low efficiency and high cost. In order to fully exploit the potential of SSRs as genetic marker, we developed a novel method for SSR genotyping, named as AmpSeq-SSR, which combines multiplexing polymerase chain reaction (PCR), targeted deep sequencing and comprehensive analysis. AmpSeq-SSR is able to genotype potentially more than a million SSRs at once using the current sequencing techniques. In the current study, we simultaneously genotyped 3105 SSRs in eight rice varieties, which were further validated experimentally. The results showed that the accuracies of AmpSeq-SSR were nearly 100 and 94% with a single base resolution for homozygous and heterozygous samples, respectively. To demonstrate the power of AmpSeq-SSR, we adopted it in two applications. The first was to construct discriminative fingerprints of the rice varieties using 3105 SSRs, which offer much greater discriminative power than the 48 SSRs commonly used for rice. The second was to map Xa21, a gene that confers persistent resistance to rice bacterial blight. We demonstrated that genome-scale fingerprints of an organism can be efficiently constructed and candidate genes, such as Xa21 in rice, can be accurately and efficiently mapped using an innovative strategy consisting of multiplexing PCR, targeted sequencing and computational analysis. While the work we present focused on rice, AmpSeq-SSR can be readily extended to animals and micro-organisms.
简单序列重复（SSR）的准确高效基因分型是SSR作为一种具有多种应用的有效遗传标记的基础。然而，现有的SSR基因分型方法存在灵敏度低、准确性低、效率低和成本高的问题。为了充分发挥SSR作为遗传标记的潜力，我们开发了一种新的SSR基因分型方法，名为AmpSeq-SSR，它结合了多重聚合酶链反应（PCR）、靶向深度测序和综合分析。使用当前的测序技术，AmpSeq-SSR能够一次性对潜在超过一百万个SSR进行基因分型。在本研究中，我们同时对八个水稻品种中的3105个SSR进行了基因分型，并通过实验进一步验证。结果表明，对于纯合和杂合样本，AmpSeq-SSR的单碱基分辨率准确率分别接近100%和94%。为了证明AmpSeq-SSR的能力，我们将其应用于两个方面。第一个是使用3105个SSR构建水稻品种的鉴别指纹图谱，其鉴别能力比通常用于水稻的48个SSR强得多。第二个是定位Xa21，一个赋予水稻对白叶枯病持久抗性的基因。我们证明了可以通过由多重PCR、靶向测序和计算分析组成的创新策略，高效构建生物体的基因组规模指纹图谱，并准确高效地定位候选基因，如水稻中的Xa21。虽然我们目前的工作聚焦于水稻，但AmpSeq-SSR可以很容易地扩展到动物和微生物。
