Isolation and characterization of hypervariable microsatellites in the red grouse Lagopus lagopus scoticus
Isolation and characterization of hypervariable microsatellites in the red grouse Lagopus lagopus scoticus
复制标题
红松鸡 Lagopus lagopus scoticus 超变微卫星的分离和表征
DOI：
10.1046/j.1365-294x.1997.00154.x
复制
发表时间：
1997
影响因子：
4.9
通讯作者：
J. Dallas
中科院分区：
文献类型：
--
作者：
S. Piertney;J. Dallas
丰度的周期性波动（Williams 1985; Moss & Watson 1991）。对这种振荡的经典解释，包括食物供应、寄生虫丰度和捕食等外在因素，已经提出（见Lance & Lawton 1990；以及其中的参考文献），尽管没有一种解释特别适用于整个种群的地理范围。最近，社会互动和亲属结构也被牵扯进来（Moss & Watson 1985; Mountford et al. 1990），守地盘的公鸡有助于将儿子和兄弟招募到群体中。检验这种“亲缘关系假说”的基础是能够准确确定单个松鸡之间的遗传亲缘关系。迄今为止，这已被证明是困难的——观察分析和标记已被证明是不确定的，而且是高度劳动密集型的。分子标记在研究其他鸟类的亲缘关系和父系关系方面是非常宝贵的（Ellegren 1992; Westneat & Webster 1994），并且在这种情况下为分析亲缘关系提供了相当大的潜力。在这里，我们描述了红松鸡中几个高变微卫星位点的分离和特征，简要讨论了这些标记如何用于亲属假设的检验，并研究了这些引物在其他镓形物种中的潜在用途。将sau3ai消化的松鸡DNA连接到λ-Zap噬菌体载体（Stratagene）上，构建了300 - 800 bp的部分基因组文库。该文库用（AC/GT）n和（AG/CT）n二核苷酸聚合物（Pharmacia）进行高强度探针检测，所得阳性转化子使用ABI 377自动测序仪进行测序（根据制造商协议进行循环测序）。PCR引物是根据微卫星的侧翼序列设计的，这些微卫星具有超过15个基序的纯阵列。使用O L I G O （version 4.1）软件（National Biosciences Inc.）设计引物，每个引物满足5个标准：(I)扩增的PCR片段总大小小于250 bp；（ii）引物GC含量为p40%；（iii）发夹形成、引物-二聚体形成和假引物电位可忽略不计；（iv） 3 '端是G、C或更好的GC；(v)引物PCR退火温度差异小于3℃，且在50 ~ 65℃范围内。根据Ellegren（1991）的说法，从一只饲养谱系的8只鸟（母亲、父亲和6只后代）的羽毛中提取了DNA，并从苏格兰东北部Glas Choille和Micras moor的50只无关鸟类的全血样本中提取了DNA（根据Bruford et al. 1992）。所有PCR扩增均采用MJ Research PTC-100热循环仪，扩增总量为10 μl。反应混合物含有10 ng模板DNA， 2.5 mM MgCl2, 75 mM Tris-HCl (pH 9.0), 20 mM (NH4)2SO4, 0.01% (v/v) twein -20，每个核苷酸0.2 mM，每个引物5 pmol（前引物末端标记32P γ (ATP)和0.5单位Taq聚合酶）。90℃初始变性3 min后，进行30个PCR循环，每个循环包括在90℃下变性30秒，在适当的退火温度下变性30秒（见表1）。除了最后一步退火后在72°C下的2分钟时间外，程序中没有延长步骤。PCR片段在6%变性聚丙烯酰胺凝胶（Sambrook et al. 1989）上电泳分离（Sambrook et al. 1989），并通过与M13mp8 DNA测序标准同时运行的比对来确定等位基因大小。在筛选的15000个微卫星重复序列中，41个（0.27%）给出了阳性信号，可以根据这10组引物进行设计。其余的包含短和（或）不完善的微卫星阵列。表1描述了红松鸡扩增微卫星位点的各种特征。所有引物对都得到了预期大小的单个产物，并且都被证明是多态性的。对已知家族群体的带带模式分析表明，这些位点的分离符合孟德尔预期。变异水平较高，平均等位基因数为9.6（±3.0）个，平均杂合度为0.77（±0.11）个。这些水平的变异会产生低概率的基因型共享和亲子关系的错误包含。因此，使用这些位点中的几个同时P R I M E R N O T E
cyclic fluctuations in abundance (Williams 1985; Moss & Watson 1991). Classic explanations for such oscillations, encompassing extrinsic factors such as food supply, parasite abundance and predation have been proposed (see Lance & Lawton 1990; and references therein), although none appear particularly appropriate over the whole geographical range of populations. More recently, social interactions and kin structure have been implicated (Moss & Watson 1985; Mountford et al. 1990), with territory-holding cocks facilitating the recruitment of sons and brothers into the population. Fundamental to testing this ‘kinship hypothesis’ is the ability to determine accurately the genetic relatedness between individual grouse. Hitherto, this has proven difficult – observational analysis and tagging has proven inconclusive as well as highly labour intensive. Molecular markers have been invaluable in studies of kinship and paternity in other avian species (Ellegren 1992; Westneat & Webster 1994), and offer considerable potential for analysis of relatedness in this case. Here we describe the isolation and characterization of several hypervariable microsatellite loci in red grouse, briefly discuss how such markers can be utilized in tests of the kinship hypothesis, and examine the potential use of these primers in other galliform species. A 300–800-bp partial genomic library was constructed by ligating Sau3AI-digested grouse DNA into a λ-Zap phagemid vector (Stratagene). This library was probed with (AC/GT)n and (AG/CT)n dinucleotide polymers (Pharmacia) at high stringency, and the resultant positive transformants sequenced using an ABI 377 automated sequencer (cycle sequencing according to manufacturers protocol). PCR primers were designed from flanking sequences for those microsatellites possessing pure arrays of more than 15 motifs. Primers were designed using O L I G O (version 4.1) software (National Biosciences Inc.), and each satisfied five criteria: (i) the total size of the amplified PCR fragment was less than 250 bp; (ii) the GC content of primers was P 40%; (iii) hairpin formation, primer–dimer formation and false priming potential was negligible; (iv) the 3’ terminus was a G, C, or better GC; (v) the difference between the PCR annealing temperatures of the primers was less than 3 °C, and within the range 50–65 °C. DNA was extracted from the feathers of eight birds from a captive pedigree (mother, father and six offspring) according to Ellegren (1991), and from whole-blood samples of 50 unrelated birds (according to Bruford et al. 1992) from Glas Choille and Micras moors in NE Scotland. All PCR amplifications were performed in a total volume of 10 μl using a MJ Research PTC-100 thermal cycler. The reaction mix contained 10 ng template DNA, 2.5 mM MgCl2, 75 mM Tris-HCl (pH 9.0), 20 mM (NH4)2SO4, 0.01% (v/v) Tween-20, 0.2 mM of each nucleotide, 5 pmol of each primer (forward primer end-labelled with 32P γ (ATP) and 0.5 units Taq polymerase. After an initial denaturation step of 3 min at 90 °C, 30 cycles of PCR were performed, each cycle consisting of 30-s denaturation at 90 °C, and 30 s at the appropriate annealing temperature (see Table 1). No extension steps were included in the programme, except for a 2-min period at 72 °C following the final annealing step. PCR fragments were resolved by electrophoresis on 6% denaturing polyacrylamide gels (Sambrook et al. 1989), and allele sizes were determined by comparison with an M13mp8 DNA sequencing standard run concurrently. Of the 15 000 transformants screened for microsatellite repeats, 41 (0.27%) gave positive signals, and from these 10 sets of primers could be designed. The remainder contained short and/or imperfect microsatellite arrays. Table 1 describes various characteristics of the amplified microsatellite loci in red grouse. All primer pairs gave a single product of expected size following PCR, and all proved polymorphic. Analysis of the banding patterns within known family groups indicate that the loci segregate according to Mendelian expectations. High levels of variability are observed with a mean allele number of 9.6 (± 3.0) and mean observed heterozygosity of 0.77 (± 0.11). These levels of variability would generate low probabilities of genotype sharing and false inclusion of parentage. As such, the use of several of these loci simultaneously P R I M E R N O T E