Reliable allele detection using SNP-based PCR primers containing Locked Nucleic Acid: application in genetic mapping.
Reliable allele detection using SNP-based PCR primers containing Locked Nucleic Acid: application in genetic mapping.
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DOI：
10.1186/1746-4811-3-2
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发表时间：
2007-02-07
期刊：
影响因子：
5.1
通讯作者：
Trognitz B
中科院分区：
文献类型：
--
作者：
Nakitandwe J;Trognitz F;Trognitz B
二倍体茄子(Solanum Caripense)是马铃薯和番茄的野生近缘种，对马铃薯晚疫病具有宝贵的抗性，我们对这种抗性的遗传基础很感兴趣。由于加勒比龙葵基因组内的遗传变异水平极低，因此不可能通过使用传统的染色体特异的SSR、RFLP、AFLP以及基因或位点特异的标记来生成密集的遗传图谱并分配单个茄子的染色体。DNA多态检测的难易程度取决于序列变异的频率和形式。近亲和近交系的狭窄遗传背景使持续的、减少的多态性的检测变得复杂，对可靠的分子标记的发展是一个挑战。然而，代表不能直接使用的常规标记的单态DNA片段在单核苷酸多态(SNPs)水平上可能包含相当大的变异。这可用于等位基因特异性分子标记的设计。基于SNPs的等位基因特异性标记的可重复性检测一直是一个技术挑战。我们提出了一种通过应用序列多态衍生(SPD)标记来检测等位基因特异性SNPs的快速且经济有效的方法。事实证明，这些标记对具有相同遗传背景的个体进行指纹识别是非常有效的。SPD标记是从非信息性的常规分子标记片段中获得的，这些标记片段被筛选为SNPs，以设计等位基因特异性的聚合酶链式反应引物。该方法利用含有单个3‘末端锁定核酸(LNA)碱基的引物。我们通过成功地进行等位基因特异的SNP标记的遗传定位，证明了该技术的适用性。这些SNP标记来自于单态保守的正交集II(COSII)标记，被定位到加勒比茄子的染色体上。通过使用SPD标记，首次可以定位16个染色体特异的COSII标记的加勒比链霉菌等位基因，并将12个连锁群中的8个分配给一致的茄子染色体。这种基于个体等位基因变异的方法允许比传统标记技术更高水平的遗传变异分辨率。我们发现，来自杂合度水平降低的生物的大多数单态分子标记片段仍然包含足以追踪单个等位基因的SNPs。
The diploid, Solanum caripense, a wild relative of potato and tomato, possesses valuable resistance to potato late blight and we are interested in the genetic base of this resistance. Due to extremely low levels of genetic variation within the S. caripense genome it proved impossible to generate a dense genetic map and to assign individual Solanum chromosomes through the use of conventional chromosome-specific SSR, RFLP, AFLP, as well as gene- or locus-specific markers. The ease of detection of DNA polymorphisms depends on both frequency and form of sequence variation. The narrow genetic background of close relatives and inbreds complicates the detection of persisting, reduced polymorphism and is a challenge to the development of reliable molecular markers. Nonetheless, monomorphic DNA fragments representing not directly usable conventional markers can contain considerable variation at the level of single nucleotide polymorphisms (SNPs). This can be used for the design of allele-specific molecular markers. The reproducible detection of allele-specific markers based on SNPs has been a technical challenge. We present a fast and cost-effective protocol for the detection of allele-specific SNPs by applying Sequence Polymorphism-Derived (SPD) markers. These markers proved highly efficient for fingerprinting of individuals possessing a homogeneous genetic background. SPD markers are obtained from within non-informative, conventional molecular marker fragments that are screened for SNPs to design allele-specific PCR primers. The method makes use of primers containing a single, 3'-terminal Locked Nucleic Acid (LNA) base. We demonstrate the applicability of the technique by successful genetic mapping of allele-specific SNP markers derived from monomorphic Conserved Ortholog Set II (COSII) markers mapped to Solanum chromosomes, in S. caripense. By using SPD markers it was possible for the first time to map the S. caripense alleles of 16 chromosome-specific COSII markers and to assign eight of the twelve linkage groups to consensus Solanum chromosomes. The method based on individual allelic variants allows for a level-of-magnitude higher resolution of genetic variation than conventional marker techniques. We show that the majority of monomorphic molecular marker fragments from organisms with reduced heterozygosity levels still contain SNPs that are sufficient to trace individual alleles.
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影响因子：
11.6
作者：
Fulton, TM;Van der Hoeven, R;Tanksley, SD
通讯作者：
Tanksley, SD
影响因子：
2.9
作者：
Koizumi, M;Morita, K;Kasuya, A
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影响因子：
3.1
作者：
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作者：
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通讯作者：
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