López-Flores Inmaculada, Ruiz-Rejón Carmelo, Cross Ismael, Rebordinos Laureana, Robles Francisca, Navajas-Pérez Rafael, de la Herrán Roberto
Departamento de Genética, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
Genetica. 2010 Dec;138(11-12):1211-9. doi: 10.1007/s10709-010-9517-1. Epub 2010 Nov 12.
When genomic DNA from the European flat oyster Ostrea edulis L. was digested by BclI enzyme, a band of about 150 bp was observed in agarose gel. After cloning and sequencing this band and analysing their molecular characteristics and genomic organization by means of Southern blot, in situ hybridisation, and polymerase chain reaction (PCR) protocols, we concluded that this band is an interspersed highly repeated DNA element, which is related in sequence to the flanking regions of (CT)-microsatellite loci of the species O. edulis and Crassostrea gigas. Furthermore, we determined that this element forms part of a longer repetitive unit of 268 bp in length that, at least in some loci, is present in more than one copy. By Southern blot hybridisation and PCR amplifications-using primers designed for conserved regions of the 150-bp BclI clones of O. edulis-we determined that this repetitive DNA family is conserved in five other oyster species (O. stentina, C. angulata, C. gigas, C. ariakensis, and C. sikamea) while it is apparently absent in C. gasar. Finally, based on the analysis of the repetitive units in these oyster species, we discuss the slow degree of concerted evolution in this interspersed repetitive DNA family and its use for phylogenetic analysis.
当用BclI酶消化欧洲扁蛎（Ostrea edulis L.）的基因组DNA时，在琼脂糖凝胶中观察到一条约150 bp的条带。对该条带进行克隆和测序，并通过Southern印迹、原位杂交和聚合酶链反应（PCR）方法分析其分子特征和基因组结构后，我们得出结论，该条带是一个散布的高度重复DNA元件，其序列与欧洲扁蛎和太平洋牡蛎（Crassostrea gigas）的（CT）-微卫星位点的侧翼区域相关。此外，我们确定该元件是一个长度为268 bp的较长重复单元的一部分，至少在某些位点，该重复单元存在多个拷贝。通过Southern印迹杂交和PCR扩增（使用针对欧洲扁蛎150 bp BclI克隆的保守区域设计的引物），我们确定这个重复DNA家族在其他五个牡蛎物种（O. stentina、C. angulata、C. gigas、C. ariakensis和C. sikamea）中是保守的，而在C. gasar中显然不存在。最后，基于对这些牡蛎物种中重复单元的分析，我们讨论了这个散布的重复DNA家族中协同进化的缓慢程度及其在系统发育分析中的应用。
