Isolation and characterization of microsatellite loci in the bearded vulture (Gypaetus barbatus) and cross-amplification in three Old World vulture species
Isolation and characterization of microsatellite loci in the bearded vulture (Gypaetus barbatus) and cross-amplification in three Old World vulture species
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DOI：
10.1046/j.1365-294x.2000.105321.x
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发表时间：
2000-12-01
影响因子：
4.9
通讯作者：
Schmid, B
中科院分区：
文献类型：
--
作者：
Gautschi, B;Tenzer, I;Schmid, B
在过去的一百年里，胡秃鹫Gypatus barbatus在欧洲遭受了种群数量的极端下降，主要是因为狩猎，但农业的变化，特别是放牧方式的变化，也导致腐肉喂食者的食物条件很差。少量种群在比利牛斯山脉、科西嘉岛和克里特岛生存下来。到20世纪初，阿尔卑斯山的人口已经完全灭绝。为了在阿尔卑斯山重新建立一个自给自足的种群，自1986年以来，已经释放了90多只幼年胡秃鹫，它们都来自圈养种群。圈养和放生种群的遗传变异量、个体之间的系谱关系以及过去(由200多个博物馆标本代表)和现在野生种群之间的基因流动程度是制定遗传管理战略的重要标准。本文介绍了用于胡子秃鹫遗传分析的14个微卫星引物的开发。我们设计了这些引物，特别强调它们后来用于古代DNA(ADNA)，并测试了它们是否适用于其他东半球秃鹫物种。我们使用改进的Tenzer等人(1999)的方法构建了一个丰富的CA和GA重复序列的基因组文库。根据Tenzer等人(1999)，用Tsp509I(New England Biolabs)消化总基因组DNA，分离200-700bp片段并连接到TSPADSHORT/TSPADLONG接头序列。该连接反应产生钝端分子，在40个25µL反应中扩增得到，其中含有10 mTris-HCl，pH 9.0；50 mKCl1，1.5 mMgCl2；0.1%TritonX 100；0.2 mg/mLBSA，每个dNTP 150µm，1µm TSPADSHORT(引物)，1.25U Taq DNA聚合酶(Appligene Oncor)。PTC100™可编程热控制器(MJ Research)上的热分布是93℃1分钟、55℃1分钟和72℃1分钟的30个循环。在72℃下，最初的5分钟延长步骤允许DNA聚合酶合成基因组DNA和连接物序列之间的缺口。聚合酶链式反应(PCR)产物与生物素化(CA)13和(GA)13探针杂交，该探针固定在法国DYNAL公司的DYNAADS M-280链霉亲和素上。按照Tenzer等人(1999)的描述进行杂交。在没有初始延伸步骤的情况下，在如上所述的第二次PCR中扩增保留片段。聚合酶链式反应产物用EcoRI(Amersham Pharmacia)消化，准备与去磷酸化的pUC18(预切EcoRI/BAP，amersham Pharmacia)连接。将JM109高效感受态细胞(Promega)转化到选择性琼脂上，将菌落斑点杂交到尼龙膜上(Hyond™-N+，amersham Pharmacia)，用ECL3‘-寡核苷酸标记和检测系统(Amersham Pharmacia)标记的寡核苷酸探针筛选含有CA和GA重复序列的插入片段。杂交是按照制造商的说明进行的。
During the last one hundred years the bearded vulture, Gypaetus barbatus, has suffered extreme population declines in Europe primarily because of hunting, but changes also in agriculture, and especially in grazing practices, have resulted in poor food conditions for carrion feeders. Small populations have survived in the Pyrenees, on Corsica and Crete. The population in the Alps was completely extinct by the beginning of the 20th century. To re-establish a self-sustaining population in the Alps, over 90 juvenile bearded vultures have been released since 1986, all originating from a captive population. The amount of genetic variability in the captive and released populations, genealogical relationships between individuals, and the degree of gene flow among wild populations in the past (represented by over 200 Museum specimens) and at present are important criteria in the development of a genetic management strategy. We describe the development of 14 microsatellite primers for conservation genetic analyses of the bearded vulture. We designed the primers with special emphasis on their later use for ancient DNA (aDNA) and tested their suitability for use in other Old World vulture species.We constructed a genomic library enriched for CA and GA repeats using a modification of the method described in Tenzer et al.(1999). Digestion of total genomic DNA with Tsp509I (New England Biolabs), isolation of 200–700 bp fragments and ligation to TSPADSHORT/TSPADLONG linker sequences were carried out according to Tenzer et al.(1999). The ligation produces blunt-ended molecules that were amplified in 40 25 µL reactions containing 10 m m Tris-HCl, pH 9.0; 50 m m KCl, 1.5 m m MgCl 2; 0.1% TritonX 100; 0.2 mg/mL BSA, 150 µ m of each dNTP, 1 µ m of TSPADSHORT (primer), and 1.25 U of Taq DNA polymerase (Appligene oncor). The thermal profile on a PTC100™ Programmable Thermal Controller (MJ Research) was 30 cycles of 93 C for 1 min, 55 C for 1 min and 72 C for 1 min. An initial 5 min extension step at 72 C allowed the DNA polymerase to synthesize the nick between genomic DNA and linker sequences. Polymerase chain reaction (PCR) products were hybridized to biotinylated (CA) 13 and (GA) 13 probes that were immobilized onto Dynabeads M-280 Streptavidin (DYNAL, France). Hybridization was carried out as described in Tenzer et al.(1999). Retained fragments were amplified in a second PCR as above without the initial extension step. PCR products were digested with EcoRI (Amersham Pharmacia) in preparation for ligation with dephosphorylated pUC18 (precut EcoRI/BAP, Amersham Pharmacia). Following transformation of JM109 High Efficiency competent cells (Promega), plating onto selective agar media and dot-blotting colonies onto Nylon-Membrane (Hybond™-N+, Amersham Pharmacia), the library was screened for inserts containing CA and GA repeats using oligonucleotide probes labelled with ECL3′-oligolabelling and detection system (Amersham Pharmacia). Hybridization was carried out in accordance with the manufacturer’s instructions.