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为了能在分子水平上有效鉴定具有粘果山羊草(Aegilops kotschyi)、偏凸山羊草(A.ventricosa)、普通小麦变种斯卑尔脱(Triticum spelta)细胞质雄性不育系及其保持系90-110和8222,提高其在杂交小麦(Triticum aestivum L.)研究与应用中的定向遗传改良,本研究对其线粒体DNA进行了扩增片段长度多态性(amplified fragment length polymorphism,AFLP)标记和序列特征性片段扩增区域(sequence characterized amplified region,SCAR)标记。通过AFLP标记方法,应用64对引物组合EcoRⅠ-NNN/MseⅠ-NNN对小麦同核异质雄性不育系和保持系进行扩增,共扩增出682条带,其中113条为多态性条带。引物E-AGG/M-CTA组合在粘果山羊草细胞质雄性不育系中扩增出一条大小约300 bp的特异性条带,对该特异条带进行回收、测序,利用Primer Premier 5.0软件重新设计SCAR引物,并对这3种类型同核异质小麦细胞质雄性不育系和保持系进行扩增,其中引物YW1在3种细胞质雄性不育系和保持系中都扩增出条带,而引物YW2仅在粘果山羊草细胞质雄性不育系扩增出一条198 bp的特异性片段,结果表明,已成功地将AFLP标记转化为操作简便、表现稳定的SCAR标记。此片段与小麦线粒体基因组有很高的同源性(同源性为99%),为烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶基因(GenBank登录号:EU534409.1)上的序列,该酶是线粒体中氧化磷酸化的入口酶,与小麦细胞质雄性不育密切相关。本研究可以用于粘类小麦细胞质雄性不育系分子标记辅助育种,也为小麦细胞质种性鉴定提供了技术支撑和理论依据。
In order to be able to identify effectively at the molecular level Aegilops kotschyi, A.ventricosa, the Triticum spelta cytoplasmic male sterile line and its maintainer line 90- 110 and 8222 to improve their directional genetic improvement in the research and application of hybrid wheat (Triticum aestivum L.). In this study, their mitochondrial DNAs were amplified by AFLP (amplified fragment length polymorphism) Sequence characterized amplified region (SCAR) markers. A total of 682 bands were amplified by AFLP markers using 64 pairs of primers EcoRⅠ-NNN / MseⅠ-NNN, and a total of 682 bands were amplified, of which 113 were polymorphic band. The primer E-AGG / M-CTA was used to amplify a specific band about 300 bp in the cytoplasmic male sterile line of Aegilops, so the specific band was recovered, sequenced and re-used by Primer Premier 5.0 software SCAR primers were designed and amplified from these three types of isoplasmic wheat cytoplasmic male sterile lines and maintainer lines. Primer YW1 amplified bands in all three cytoplasmic male sterile lines and maintainer lines. However, The primer YW2 only amplified a 198 bp specific fragment in the Aegilops sinensis cytoplasm male sterile line. The results showed that the AFLP marker has been successfully transformed into a simple and stable SCAR marker. This fragment has high homology with the wheat mitochondrial genome (99% homology) and is a sequence on the NADH dehydrogenase gene (GenBank accession number: EU534409.1) , Which is the oxidative phosphorylation entry enzyme in mitochondria, is closely related to the cytoplasmic male sterility of wheat. This study can be used for molecular marker-assisted breeding of CMS lines in wheat and provides technical support and theoretical basis for the identification of wheat cytoplasm.