Chérif Mohamed, Chilvers Martin I, Akamatsu Hajime, Peever Tobin L, Kaiser Walter J
Laboratoire de Phytopathologie, Institut National Agronomique de Tunisie, Cité Mahrajéne, Tunis, Tunisia.
Curr Genet. 2006 Sep;50(3):203-15. doi: 10.1007/s00294-006-0085-y. Epub 2006 Jul 18.
The mating type (MAT) locus of the lentil pathogen, Ascochyta lentis, was cloned and characterized using thermal asymmetric interlaced and inverse PCR with primers designed to the HMG-box of Ascochyta rabiei. A multiplex PCR assay for mating type was developed based on MAT idiomorph and flanking sequences. Primers were designed to specifically amplify MAT from several Ascochyta spp. including A. pisi, A. fabae and A. viciae-villosae in addition to A. lentis. Four hundred and fifty and 700 bp fragments were amplified from MAT1-1 and MAT1-2 isolates, respectively, and fragment size correlated perfectly with laboratory crosses using mating type tester strains. MAT-specific PCR allowed rapid scoring of mating type in crude DNA extracts from geographically diverse population samples of A. viciae-villosae from California and Washington State, USA. This co-dominant MAT-specific PCR assay will be a valuable tool for studying the population structure, biology and epidemiology of these fungi.
使用热不对称交错PCR和反向PCR，以针对鹰嘴豆壳二孢（Ascochyta rabiei）HMG框设计的引物，克隆并表征了小扁豆病原体扁豆壳二孢（Ascochyta lentis）的交配型（MAT）位点。基于MAT特异性等位基因及其侧翼序列，开发了一种用于交配型的多重PCR检测方法。除了扁豆壳二孢外，还设计了引物以特异性扩增来自几种壳二孢属物种（包括豌豆壳二孢、蚕豆壳二孢和绒毛野豌豆壳二孢）的MAT。分别从MAT1-1和MAT1-2分离株中扩增出450 bp和700 bp的片段，片段大小与使用交配型测试菌株进行的实验室杂交结果完全相关。MAT特异性PCR能够快速确定来自美国加利福尼亚州和华盛顿州地理分布多样的绒毛野豌豆壳二孢群体样本粗DNA提取物中的交配型。这种共显性的MAT特异性PCR检测方法将成为研究这些真菌群体结构、生物学和流行病学的有价值工具。
