Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction:: its application to mapping and gene tagging in Brassica
Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction:: its application to mapping and gene tagging in Brassica
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DOI：
10.1007/s001220100570
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发表时间：
2001-08-01
影响因子：
5.4
通讯作者：
Quiros, CF
中科院分区：
文献类型：
--
作者：
Li, G;Quiros, CF
我们开发了一种简单的标记技术，称为序列相关扩增多态性（SRAP），旨在扩增开放阅读框（ORF）。它基于双引物扩增。引物长17或18个核苷酸，由以下元件组成。核心序列长13至14个碱基，其中起始于5'端的前10或11个碱基是无特定结构的序列（“填充物”序列），随后是正向引物中的序列CCGG和反向引物中的序列AATT。核心之后是3'端的三个选择性核苷酸。正向和反向引物的填充序列必须彼此不同，并且可以是10或11个碱基长。对于前五个循环，退火温度设定为35 ℃。以下35个循环在50 ℃下进行。扩增的DNA片段通过变性丙烯酰胺凝胶分离并通过放射自显影检测。我们在一系列甘蓝重组自交系和双单倍体材料上进行了标记技术的试验。测序后，大约45%的凝胶分离条带与Genbank数据库中的已知基因相匹配。测序结果表明，20%的SRAP标记为共显性。构建的连锁图显示，平均分布的SRAP标记在9个主要的连锁群，在这方面没有不同的AFLP标记。我们成功地用这些标记物标记了硫代葡萄糖苷去饱和基因BoGLS-ALK。SRAP在其他作物如马铃薯、水稻、莴苣、大白菜（Brassica rapa L.）油菜（Brassica napus L.），大蒜苹果柑橘和芹菜我们还扩增了从大白菜不同组织中分离的cDNA，允许这些序列的指纹图谱。
We developed a simple marker technique called sequence-related amplified polymorphism (SRAP) aimed for the amplification of open reading frames (ORFs). It is based on two-primer amplification. The primers are 17 or 18 nucleotides long and consist of the following elements. Core sequences, which are 13, to 14 bases long, where the first 10 or 11 bases starting at the 5' end, are sequences of no specific constitution ("filler" sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. The core is followed by three selective nucleotides at the 3' end. The filler sequences of the forward and reverse primers must be different from each other and can be 10 or 11 bases long. For the first five cycles the annealing temperature is set at 35 degreesC. The following 35 cycles are run at 50 degreesC. The amplified DNA fragments are separated by denaturing acrylamide gels and detected by autoradiography. We tested the marker technique in a series of recombinant inbred and doubled-haploid lines of Brassica oleracea L. After sequencing, approximately 45% of the gel-isolated bands matched known genes in the Genbank database. Twenty percent of the SRAP markers were co-dominant, which was demonstrated by sequencing. Construction of a linkage map revealed an even distribution of the SRAP markers in nine major linkage groups, not differing in this regard to AFLP markers. We successfully tagged the glucosinolate desaturation gene BoGLS-ALK with these markers. SRAPs were also easily amplified in other crops such as potato, rice, lettuce, Chinese cabbage (Brassica rapa L.), rapeseed (Brassica napus L.), garlic, apple, citrus, and celery. We also amplified cDNA isolated from different tissues of Chinese cabbage, allowing the fingerprinting of these sequences.