鸻形目鸟类线粒体基因组测序策略

Strategy of Amplification and Sequencing of the Mitochondrial Genome of Charadriiformes

鸻形目(Charadriiformes),全世界约有384个物种,分属于19科94属,种类繁多、分布广泛,是研究迁徙和觅食行为的良好材料。近年来,线粒体基因组的研究快速发展,由于样品难以收集,缺乏系统的测序策略,鸻形目鸟类线粒体基因组的研究相对滞后。引物设计对聚合酶链式反应(PCR)至关重要,一个成功的PCR实验依赖于高质量的特异性引物,本研究拟设计一套用于鸻形目鸟类线粒体基因组扩增的通用引物。对GenBank中现有的鸻形目物种线粒体基因组进行多重比对,发现若干个保守性区域,本研究在该区域设计13对扩增鸻形目鸟类线粒体基因组的通用引物,扩增的目的片段长度均在1.5 kb左右。我们选取4个物种,即灰头麦鸡(Vanellus cinereus)、丘鹬(Scolopax rusticola)、白腰草鹬(Tringaochropus)和针尾沙锥(Gallinagostenura),进行PCR扩增验证,设计的引物在4个物种中均能顺利扩增、测序,该引物在鸻形目鸟类线粒体基因组扩增中的具有普遍适用性。本研究设计的13对通用引物在扩增鸻形目物种线粒体全基因组中具有较强的应用价值,将为鸻形目的系统发育关系、种群遗传学和生物地理学研究提供珍贵的资源。

Charadriiformes（Vertebrate： Aves） is a species-rich order with approximately 384 species belonging to 94 genera of 19 families（Fig. 1）, and contains some model species to study migration and foraging behavior. The resources of mitogenome have rapidly accumulated in recent years due to the advanced genomic sequencing, however, Charadriiformes mitogenome has not been well studied（Table 2）. Primer design has crucial importance to PCR experiments, and a successful PCR experiment depends on the quality and specificity of the primers. After the multiple sequence alignment, we found many conserved regions. Then, we designed the forward and reverse primers according to the overlapping and conserved regions. In this study, we designed 13 pairs of primers, with the amplified target fragment for each pair about 1.5 kb in length（Fig. 2, Table 1）. In order to test the proposed method and prove the performance of these primer pairs, the whole primer settings were applied to amplify and sequence the mitochondrial genomes of four birds： Grey-headed Lapwing Vanellus cinereus, Eurasian Woodcock Scolopax rusticola, Green Sandpiper Tringa ochropus, and Pin-tailed Snipe Gallinago stenura（Fig. 3）. The results proved that these primer pairs were efficient. Taken together, the designed primer pairs are an effective toolkit of amplification and sequencing for the Charadriiformes mitogenomes. Our work provides a valuable resource facilitating further study of molecular systematics, population genetics, and phylogeography of Charadriiformes.