Isolation and characterization of microsatellite loci in the cichlid fish Pseudotropheus zebra
Isolation and characterization of microsatellite loci in the cichlid fish Pseudotropheus zebra
复制标题
丽鱼科鱼 Pseudotropeus zebra 微卫星位点的分离和表征
作者：
M. J. H. Oppen;C. Rico;J. Deutsch;G. Turner;G. Hewitt
和(CT/GA)n个微卫星基因座，目的是建立一种聚合酶链式反应系统来分析A.L.F.测序仪(Pharmacia)上的单个基因座。按照Rico等人的描述构建了大小选择的基因组文库。(1994b)使用来自马拉维湖恩哈塔湾的20个无关个体的DNA混合。在≈4500重组子中有42个GT和17个CT阳性，这表明在斑马基因组中大约每35kb就有一个GT重复，大约每85kb就有一个CT重复。我们已经对这些克隆中的45个进行了测序，如Rico等人所述。(1994a)，并用O L I G O TM Macintosh version4.0(美国国家生物科学研究所)软件为9个克隆设计了两侧区域的引物。优化了9个基因座中的7个的聚合酶链式反应条件(表1)。11-μL反应包括1个μL模板DNA(≈20 ng)，每个引物的1，1-μM(前向引物是荧光素标记的)，每个dNTP的2 0 0μM，1或3 mM的氯化镁(表1)，0.2μg牛血清白蛋白，1×NH4反应缓冲液(Bioline，英国，伦敦)和0.2 5个单位的BioTaq(Bioline)。混合物上覆盖着10μ的L矿物油。Omnigene热循环仪(HYBAID)的扩增图谱如下：初始变性步骤为94°C 3min，随后是94°C 30 S、72°C S和30 S的7个循环，然后是89°C 30 S、30 S(表1)和72°C 30 S的23个循环。扩增产物用稀释缓冲液(由600μL葡聚糖蓝负载染料[6 mg葡聚糖蓝/毫升去离子甲酰胺]和900μL去离子甲酰胺组成)稀释至适当浓度(0~12×稀释)，与内部大小标记混合后进行电泳。大小标记如下：以100 ng M13mp18+为DNA模板，使用荧光素标记的通用引物和未标记的自行设计的反向引物(表2)。对100个μ的L反应进行扩增，每个引物含有0.5μM，每个dNTP含有200μM，1×KCl2反应缓冲液中含有氯化镁(Bioline)和1.25U的BioTaq。用一滴矿物油覆盖反应混合物，并在Perkin Elmer CETUS热循环仪中进行放大，在94℃的初始变性步骤为3分钟，然后是28次循环，分别为94℃1分钟、适当的热处理温度1分钟(表2)和72℃1分钟。最后一次循环的延长时间为5分钟，而不是1分钟。然后在1%TbE-琼脂糖凝胶上进行聚合酶链式反应，用EtBR染色，从凝胶中切下正确大小的条带。将切下的凝胶放入一个穿孔的0.5μL微离聚管中，管中填满棉花。然后将试管放入1.5μ的L微离聚管中，在微离心机中旋转2-5分钟。缓冲液中的DNA是从1.5-μL试管的底部收集的，而琼脂糖留在了棉花中。用1微升该DNA进行再扩增，扩增步骤与以前相同，只是步骤缩短到30 S，而不是1分钟。在0.5~1μ范围内，对扩增产物中的L进行等位基因分型。用Sequagel Extended(National Diagnostics)在1000V、60 mA、50W、48°C和1.25 S采样时间下，在短的A.L.F.平板上进行扩增产物(两个基因座在可能的情况下合并)和大小标记。一种凝胶连续使用三次。运行时间从30分钟到55分钟不等，这取决于等位基因的大小。使用软件程序Fragment Manager TM Version 1.2(Pharmacia)确定等位基因的大小。等位基因数量和预期杂合度在不同的基因座之间差异很大(表1)。正如预期的那样，最长的重复序列是最多态的。重复序列<；7是单态的。大多数多态基因座在决定种群结构方面是有用的(M.J.H.van Oppen等人)。不公开。数据)。其他小组最近还开发了几个额外的慈菇微卫星引物(Kellogg等人)。1995年；Lee&Kocher 1996；Parker&Kornfield 1996；ZARDOYA等人。1996年)。P R I M E R N O T E
and (CT/GA)n microsatellite loci from the malawian cichlid fish Pseudotropheus zebra BB (Boulenger), with the aim of developing a PCR system to analyse individual loci on the A.L.F. sequencer (Pharmacia). A size-selected genomic library was constructed as described in Rico et al. (1994b) using a DNA mix from 20 unrelated individuals from Nkhata Bay, Lake Malawi. Amongst ≈ 4500 recombinants were 42 GT and 17 CT positives, suggesting that a GT repeat occurs roughly every 35 kb and a CT repeat roughly every 85 kb in the P. zebra genome. We have sequenced 45 of these clones as described in Rico et al. (1994a) and designed primers from both flanking regions for nine clones using the software program O L I G O TM Macintosh version 4.0 (National Biosciences). PCR conditions were optimized for seven of the nine loci (Table 1). PCR was carried out in 11-μL reactions consisting of 1 μL template DNA (≈ 20 ng), 1.1-μM of each primer (the forward primer was fluorescein-labelled), 200-μM of each dNTP, 1 or 3-mM MgCl2 (Table 1), 0.2 μg BSA, 1 × NH4 reaction buffer (Bioline, London, UK) and 0.25 units of BioTaq (Bioline). The mixture was overlaid with 10 μl mineral oil. The PCR profile used on an OmniGene Thermal Cycler (Hybaid) was as follows: an initial denaturing step of 3 min 94 °C was followed by 7 cycles of 30 s at 94 °C, 30 s at the appropriate annealing temperature (Table 1) and 30 s at 72 °C, followed by 23 cycles of 30 s at 89 °C, 30 s at the appropriate annealing temperature (Table 1) and 30 s at 72 °C. With this PCR profile genomic DNA and PCR products will be denatured and amplified during the first seven cycles, while mainly PCR products will be denatured and reamplified during the subsequent 23 cycles, resulting in fewer non-specific products. PCR products were diluted to the appropriate concentration (0–12× diluted) with dilution buffer (consisting of 600 μL Dextran-Blue loading dye [6 mg of Dextran-Blue/mL deionised formamide] and 900 μL of deionised formamide) and mixed with internal size markers prior to electrophoresis. Size markers were made as follows: 100 ng of M13mp18 + was used as DNA template using fluorescein-labelled universal primer and an unlabelled self-designed reverse primer (Table 2). PCR was carried out in 100 μL reactions, containing 0.5 μM of each primer, 200 μM of each dNTP, 1 × KCl reaction buffer containing MgCl2 (Bioline) and 1.25 units of BioTaq. The reaction mixture was overlaid with a drop of mineral oil and amplified in a Perkin Elmer Cetus thermocycler using an initial denaturing step of 3 min at 94 °C followed by 28 cycles of 1 min at 94 °C, 1 min at the appropriate annealing temperature (Table 2) and 1 min at 72 °C. The last cycle had a 5 min instead of a 1 min extension time. PCR products were then run on 1% TBE-agarose gels stained with EtBr and the band of the correct size was excised from the gel. The piece of excised gel was placed in a punctured 0.5μl microfuge tube filled with cotton wool. The tube was then placed in a 1.5 μL microfuge tube and spun in a microcentrifuge for 2–5 min. The DNA in TBE buffer was collected from the bottom of the 1.5-μL tube while the agarose remained in the cotton wool. One microlitre of this DNA was used for reamplification using the same PCR profile as before except that the steps were shortened to 30 s instead of 1 min. Between 0.5 and 1 μL of these PCR products was used to size the various alleles. PCR products (two loci were combined where possible) and size markers were run on short A.L.F. plates using Sequagel Extended (National Diagnostics) at 1000 V, 60 mA, 50 W, 48 °C and 1.25 s sampling time. A single gel was used for three consecutive runs. Running time varied between 30 and 55 min, depending on the size of the alleles. Alleles were sized using the software program Fragment ManagerTM version 1.2 (Pharmacia). Number of alleles and expected heterozygosities varied considerably among the different loci (Table 1). As expected, the longest repeats were most polymorphic. Repeats < 7 were monomorphic. Most of the polymorphic loci are useful in resolving population structure (M. J. H. van Oppen et al. unpubl. data). Several additional cichlid microsatellite primers have recently been developed by other groups (Kellogg et al. 1995; Lee & Kocher 1996; Parker & Kornfield 1996; Zardoya et al. 1996). P R I M E R N O T E