Gonzalez P, Labarère J
Laboratory of Molecular Genetics and Breeding of Cultivated Mushrooms, INRA-University Victor Segalen Bordeaux 2, 33883 Villenave d'Ornon Cédex, France.
Appl Environ Microbiol. 1998 Nov;64(11):4149-60. doi: 10.1128/AEM.64.11.4149-4160.1998.
A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The conservation of the sequences of the V4, V6, and V9 domains of the mitochondrial SSU rRNA within species and the high degree of interspecific variation found in the Agrocybe species studied open the way for these sequences to be used as specific molecular markers of the Basidiomycota.
对侧耳属进行了线粒体小亚基（SSU）rRNA可变结构域V4、V6和V9的比较研究，通过PCR扩增了属于10个物种的42个野生分离株，这些物种包括高大环柄菇、硬柄环柄菇、茶薪菇、埃氏环柄菇、坚固环柄菇、早熟环柄菇、沼生环柄菇、小孢环柄菇、桤木环柄菇和韦氏环柄菇。PCR产物测序表明，属于同一物种的分离株中这三个结构域长度相同且序列相同，而在这10个物种之间发现了变异。序列比对显示，在每个可变结构域最小序列中遇到的核苷酸基序在最大序列中也能找到，这表明序列是通过插入-缺失事件进化而来的。每个结构域二级结构的测定表明，插入-缺失事件通常发生在不直接参与二级结构的区域（即环）。此外，在每个结构域的起始和末端发现了长度为4至25个核苷酸的保守序列，这些序列可能构成属特异性序列。对V4、V6和V9二级结构的比较导致识别出以下四组：（i）第一组，其特征是V4结构域中存在额外的P23-1和P23-3螺旋，V9中缺乏P49-1螺旋，包括高大环柄菇、茶薪菇和埃氏环柄菇；（ii）第二组，其V4中有P23-3螺旋，V9中有P49-1螺旋，包括小孢环柄菇；（iii）第三组，其V4中没有额外的螺旋，V9中有P49-1螺旋，包括沼生环柄菇、坚固环柄菇、桤木环柄菇和早熟环柄菇；（iv）第四组，其V4中缺乏额外的螺旋且V9中缺乏P49-1螺旋，包括韦氏环柄菇和硬柄环柄菇。基于可变结构域序列的共识树结构支持了物种的这种分组。线粒体SSU rRNA的V4、V6和V9结构域序列在物种内的保守性以及在所研究的侧耳属物种中发现的高度种间变异，为将这些序列用作担子菌门的特异性分子标记开辟了道路。
