Identification of S-alleles using polymerase chain reaction-cleaved amplified polymorphic sequence of the S-locus receptor kinase in inbreeding lines of Brassica oleracea
Identification of S-alleles using polymerase chain reaction-cleaved amplified polymorphic sequence of the S-locus receptor kinase in inbreeding lines of Brassica oleracea
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DOI：
10.1046/j.1439-0523.2002.00713.x
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发表时间：
2002-06-01
期刊：
影响因子：
2
通讯作者：
Nou, IS
中科院分区：
文献类型：
--
作者：
Park, JI;Lee, SS;Nou, IS
采用花粉管通道试验、聚合酶链反应-切割扩增多态性序列（PCR-CAPS）和核苷酸序列测定等方法对S位点受体激酶基因（SRK）进行了鉴定和DNA多态性分析。SRK特异性引物可以区分I类和II类S单倍型，扩增900-1050 bp的单个DNA片段。用SRK Ⅰ类特异引物对22个自交系的DNA片段进行扩增，用Hin fI和Eco RII鉴定出7个类型。此外，用SRK Ⅱ类特异引物对17个自交系的DNA片段进行扩增，确定了Alu Ⅰ的3种类型。对10个S单倍型的SRK基因扩增片段进行序列测定，结果表明SRK基因3 '端外显子高度保守，内含子变异较大，导致PCR-CAPS图谱的带型具有多态性。通过PCR产物的限制性酶切分析确定了植株的S单倍型，并与基于花粉管生长试验的结果一致。利用SRK特异性引物对进行的PCR-CAPS分析可用于育种中S等位基因的鉴定。
Identification and DNA polymorphism of the S -locus receptor kinase gene (SRK ) was analysed by pollen tube tests, polymerase chain reaction-cleaved amplified polymorphic sequence (PCR-CAPS) and nucleotide sequencing. SRK -specific primers that can distinguish class I and class II S haplotypes amplified single DNA fragments of 900-1050 bp. The DNA fragments of 22 inbred lines amplified with a class I SRK -specific primer pair determined seven types with Hin fI and Eco RII. In addition, the DNA fragments of 17 inbred lines amplified with a class II SRK -specific primer pair determined three types with Alu I. Nucleotide sequencing of the DNA fragments amplified from 10 S haplotypes showed that exons of the 3'-end in SRK are highly conserved, and that there is much variation of the introns, which produced polymorphism of the band pattern in PCR-CAPS profiles. The S haplotypes of the plants were determined by restriction analysis of PCR products and agreed with results based on pollen tube growth tests. The PCR-CAPS analysis using specific primer pairs of SRK is considered to be useful for S allele identification in breeding programmes.