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用于最大化植物育种中高分辨率熔解分析稳健性的SNP位点选择标准。

Selection criteria for SNP loci to maximize robustness of high-resolution melting analysis for plant breeding.

作者信息

Yamagata Yoshiyuki, Yoshimura Atsushi, Anai Toyoaki, Watanabe Satoshi

机构信息

Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi, Fukuoka 819-0395, Japan.

Faculty of Agriculture, Saga University, 1 Honjo-machi, Saga, Saga 840-8502, Japan.

出版信息

Breed Sci. 2018 Sep;68(4):488-498. doi: 10.1270/jsbbs.18048. Epub 2018 Sep 4.

DOI:10.1270/jsbbs.18048
PMID:30369824
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6198901/
Abstract

DNA markers are useful for identifying genes and developing new genetic materials for breeding and genetic research. High-resolution melting (HRM) analysis can detect a single nucleotide polymorphism (SNP) in two polymerase chain reaction (PCR) fragments as a melting temperature (Tm) difference without additional experimental steps, such as gel electrophoresis. To design a method for developing reliable HRM markers that discriminate between homozygous alleles containing SNPs, we tested new evaluation indexes related to the thermodynamics of double-stranded DNA to find one that maximizes the difference in Tm values between PCR fragments. We found that differences in the change in Gibbs free energy (Δ) correlated with actual differences in Tm values. Optimization of the nearest neighboring nucleotide (NNN) of a SNP by nucleotide substitution in the primer and reducing the size of the PCR fragment both enlarged the actual differences in Tm. The genetic DNA markers we developed by NNN substitution, termed NNNs-HRM markers, could be precisely mapped within soybean chromosomes by linkage analysis. We developed a Perl script pipeline to enable the automatic design of a massive number of NNNs-HRM markers; these scripts are freely available and would be useful for practical breeding programs for other plant species.

摘要

DNA标记对于识别基因以及开发用于育种和遗传研究的新遗传材料很有用。高分辨率熔解（HRM）分析可以在两个聚合酶链反应（PCR）片段中检测单核苷酸多态性（SNP），将其作为熔解温度（Tm）差异，而无需额外的实验步骤，如凝胶电泳。为了设计一种开发可靠的HRM标记的方法，以区分含有SNP的纯合等位基因，我们测试了与双链DNA热力学相关的新评估指标，以找到一个能使PCR片段之间Tm值差异最大化的指标。我们发现吉布斯自由能变化（Δ）的差异与Tm值的实际差异相关。通过在引物中进行核苷酸替换来优化SNP的最近邻核苷酸（NNN）以及减小PCR片段的大小，都扩大了Tm的实际差异。我们通过NNN替换开发的遗传DNA标记，称为NNNs-HRM标记，可以通过连锁分析精确地定位在大豆染色体上。我们开发了一个Perl脚本管道，以实现大量NNNs-HRM标记的自动设计；这些脚本可免费获取，对其他植物物种的实际育种计划将很有用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/87701ef733fd/68_18048_7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/55ccd466020c/68_18048_1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/d5f61b221bac/68_18048_2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/041be98faf20/68_18048_3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/4cf48882a3a1/68_18048_4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/2c0e12d9d409/68_18048_5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/83610554a24d/68_18048_6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/87701ef733fd/68_18048_7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/55ccd466020c/68_18048_1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/d5f61b221bac/68_18048_2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/041be98faf20/68_18048_3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/4cf48882a3a1/68_18048_4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/2c0e12d9d409/68_18048_5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/83610554a24d/68_18048_6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6f53/6198901/87701ef733fd/68_18048_7.jpg

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