A simple allele-specific PCR marker for identifying male-sterile trees: Towards DNA marker-assisted selection in the Cryptomeria japonica breeding program
A simple allele-specific PCR marker for identifying male-sterile trees: Towards DNA marker-assisted selection in the Cryptomeria japonica breeding program
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DOI：
10.1007/s11295-014-0743-z
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发表时间：
2014-08-01
影响因子：
2.4
通讯作者：
Tsumura, Yoshihiko
中科院分区：
文献类型：
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作者：
Moriguchi, Yoshinari;Ueno, Saneyoshi;Tsumura, Yoshihiko
自 20 世纪 70 年代以来，随着柳杉种植面积的增加，日本患有柳杉花粉病的人数大幅增加。为了减少花粉散布量，已经实施了使用具有雄性不育基因的树木的育种计划。我们在四个粳稻科中构建了围绕雄性不育基因（ms-1）的部分连锁图，以促进这一过程。与 ms-1 连锁最密切的标记在四个作图家族中有所不同：TO13S 家族中的 gSNP00438、gSNP01452、estSNP00083 和 estSNP01228（距离 ms-1 3.1 cM）； S3T67 家族中的 gSNP05835 和 gSNP06239（距离 ms-1 2.0 cM）； F1N4 家族中的 gSNP05835（距离 ms-1 1.5 cM）；和 T5 家族中的 gSNP06239（距离 ms-1 4.2 cM）。这可能主要是由于用于绘制家庭图谱的父母之间的遗传差异所致。然而，在所有四个科中，使用最接近的标记来识别雄性不育树的准确度都超过 96.0%。这些结果表明，使用最接近 ms-1 基因座的侧翼标记，对给定科内的雄性不育树进行标记辅助选择是可行的。我们还开发了一种等位基因特异性 PCR 标记，用于鉴定 TO13S 家族中的雄性不育树，从中产生雄性不育幼苗。使用三种引物组合的等位基因特异性 PCR 产生了两个清晰的片段，可以通过琼脂糖凝胶电泳轻松分离：一个片段的分子量为 410 bp，它存在于所有样品中，因此可以用作阳性对照；另一个片段的分子量较低（196 bp），它对雄性不育树具有特异性。该标记使得可以进行简单且经济的 PCR 测定来检测与靶基因连接的 SNP，而无需使用荧光标记。这项研究展示了如何利用高密度连锁图谱的信息来开发森林树种中重要主基因的简单等位基因特异性 PCR 标记。此外，我们的结果将有助于MAS（标记辅助选择）在针叶树中的首次应用，因为粳稻雄性不育具有多种优势，可能是MAS在针叶树中的最佳例子之一。
The number of people in Japan suffering from Cryptomeria japonica pollinosis has risen considerably since the 1970s as the area planted with this species has increased. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. We have constructed partial linkage maps surrounding a male sterility gene (ms-1) in four families of C. japonica to facilitate this process. The marker most closely linked to ms-1 was different in the four mapping families: gSNP00438, gSNP01452, estSNP00083, and estSNP01228 in the TO13S family (3.1 cM from ms-1); gSNP05835 and gSNP06239 in the S3T67 family (2.0 cM from ms-1); gSNP05835 in the F1N4 family (1.5 cM from ms-1); and gSNP06239 in the T5 family (4.2 cM from ms-1). This is probably mainly due to genetic differences between the parents used to produce the mapping families. However, in all four families, the accuracy with which male-sterile trees could be identified using the closest markers was more than 96.0 %. These results suggested that marker-assisted selection of male-sterile trees within a given family is feasible using the closest flanking markers to the ms-1 locus. We also developed an allele-specific PCR marker for identifying male-sterile trees in the TO13S family from which male-sterile seedlings are produced. Allele-specific PCR using three primer combinations produced two clear fragments, which could be easily separated by agarose gel electrophoresis: one fragment with a molecular weight of 410 bp, which was present in all samples and could thus be used as a positive control, and another of lower molecular weight (196 bp), which was specific for male-sterile trees. This marker makes it possible to carry out a simple and economical PCR assay for the detection of the SNP linked to the target gene without the need to use fluorescent labels. This study shows how a simple allele-specific PCR marker for an important major gene in a forest tree species can be developed using information from a high-density linkage map. In addition, our results will facilitate the first application of MAS (marker assisted selection) in conifers because the male sterility in C. japonica has several advantages and may be one of the best examples for MAS in conifers.