Valicente Fernando Hercos, da Silva Rosane Bezerra
Embrapa Milho e Sorgo, Rod. MG 424 KM 65, CEP 35701-970 Sete Lagoas, MG Brazil.
Department of Biotechnology, Campus Universitário, CEP 37200-000 Lavras, MG Brazil.
Springerplus. 2014 May 20;3:256. doi: 10.1186/2193-1801-3-256. eCollection 2014.
A total of 65 Bacillus thuringiensis (Bt) isolates were subjected to analysis of genetic relationship using fAFLP (fluorescent Fragment Length Polymorphism), in order to determine the genetic diversity within a group of Bt strains. 26 strains from different subspecies were identified as it follows: 9 kindly provided by the USDA (United States Department of Agriculture), 9 kindly provided by the Institute Pasteur and eight from Embrapa Maize and Sorghum Bt Collection, and 39 strains with no subspecies information also from Embrapa's Bt Collection. DNA sample was double digested with restriction enzymes EcoRI and MseI, and the fragments were linked to adapters. Selective amplification reactions were performed using five primer combinations and the amplified fragments were separated by gel electrophoresis on an ABI377 sequencer. Genetic distances were obtained by the complement of the Jaccard coefficient and the groups were performed by the UPGMA method. Five primer combinations generated 495 scorable fragments and 483 were found to be polymorphic. Out of 26 subspecies, strains 344 and T09 (B. thuringiensis subsp. tolworthi) showed the highest similarity (15%), while isolates HD3 B. thuringiensis subsp finitimus and T24 B. thuringiensis subsp neoleonensis were the most genetically distant (92%). B. thuringiensis isolates with no subspecies identification, found in samples from Goiás State showed higher similarity forming a group with an average distance of 6%, and the closest subspecies to this group was B. thuringiensis subsp thuringiensis (HD2) with 52% of similarity. This similarity may be due to the fact that these organism exchange genetic material by conjugation, and it is relatively common to have evolutionary characteristics of their ancestors.
总共65株苏云金芽孢杆菌（Bt）菌株通过荧光扩增片段长度多态性分析（fAFLP）进行遗传关系分析，以确定一组Bt菌株内的遗传多样性。鉴定出26株来自不同亚种的菌株，具体如下：9株由美国农业部（USDA）友好提供，9株由巴斯德研究所友好提供，8株来自巴西农牧业研究公司玉米和高粱Bt菌种保藏中心，还有39株无亚种信息的菌株也来自巴西农牧业研究公司的Bt菌种保藏中心。DNA样本用限制性内切酶EcoRI和MseI进行双酶切，片段与接头连接。使用五种引物组合进行选择性扩增反应，扩增片段在ABI377测序仪上通过凝胶电泳分离。通过杰卡德系数的互补得到遗传距离，并采用非加权组平均法（UPGMA）进行分组。五种引物组合产生了495个可计分片段，其中483个为多态性片段。在26个亚种中，菌株344和T09（苏云金芽孢杆菌托氏亚种）相似度最高（15%），而苏云金芽孢杆菌有限亚种HD3和苏云金芽孢杆菌新莱昂西斯亚种T24遗传距离最远（92%）。在戈亚斯州样本中发现的未鉴定亚种的苏云金芽孢杆菌菌株相似度更高，形成一个平均距离为6%的组，与该组最接近的亚种是苏云金芽孢杆菌苏云金亚种（HD2），相似度为52%。这种相似性可能是由于这些生物体通过接合交换遗传物质，并且具有其祖先的进化特征相对常见。
