防檢疫重要性薊馬（纓翅目：薊馬科）專一性引子之鑑定應用

薊馬分布廣泛，危害多種經濟作物，是各國植物防檢疫工作上的重要標的害蟲；但其個體細小，特徵細微或有種內變異，常造成分類鑑定上的困擾。藉由薊馬DNA分子研究，解決了許多分類種群的困擾，也證明一些薊馬物種具有不同的遺傳支系，無法以形態特徵區辨。本研究應用種間區別性較明確的核醣體第二區間(Internal transcribed spacer 2, ITS2) DNA片段，進行蔥薊馬(Thrips tabaci Lindeman, 1889)、南黃薊馬(Thrips palmi Karny, 1925)、梳缺花薊馬(Frankliniella schultzei (Trybom, 1910))及玉米薊馬(Frankliniella williamsi Hood, 1915)等農作上常見薊馬的專一性引子分子鑑定；擴增的ITS2長度約為500 bp，四種薊馬間ITS2平均變異範圍為40.6～44.4%；比對世界各地族群的序列發現，蔥薊馬、南黃薊馬及梳缺花薊都有高達18%以上的種內變異存在。進一步針對四種薊馬設計多組ITS2的專一性引子對，共選用15屬21種薊馬測試，各薊馬的專一性片段均可複製成功；其它種薊馬的穩定性測試，則未複製出DNA條帶。然而，比對各種薊馬ITS序列後發現，雖然多數的專一性引子序列與世界各地族群的序列一致，最多差2個鹼基位，但有2條設計的專一性引子差異高達6個鹼基位；因此，若鑑定樣本來自遺傳變異大的族群，這些專一性引子的分子鑑定應用，可能造成偽陰性偵測結果。

關鍵字

薊馬；分子鑑定；核醣體第二區間；專一性引子

並列摘要

Thrips (Thysanoptera: Thripidae) are major pests of agricultural crops worldwide. However, their minute size, highly diverse morphologies, and lack of easily recognizable characteristics often make their identification difficult. According to molecular analyses, which can provide reliable identification of species complex, numerous thrips species exhibit largely differ in their genetic composition; however, their morphological characters remain indistinguishable. In this study, sequences of the nuclear internal transcribed spacer 2 (ITS2) of agronomically damaged thrips-namely Thrips tabaci, Thrips palmi, Frankliniella schultzei, and Frankliniella williamsi-were analyzed to design species-specific primers for their molecular identification. The ITS2 fragments obtained using universal primers were approximately 500 bp in length, and the average sequence variation among the four species ranged from 40.6% to 44.4%. With sequences for T. tabaci, T. palmi, and F. schultzei from GenBank included in the analysis, more than 18% intraspecific sequence variation was found. ITS2 sequences were employed to design multiple sets of species-specific primers of the four thrips species. Examination of primer specificity and stability in 21 thrips species of 15 genera revealed the expected amplified products in the four target species, with no cross-amplifications. Sequence alignment of the specific primers with other conspecific GenBank sequences showed that these primers had conserved sequences with a mismatch of less than two nucleotides. However, more than six nucleotide mismatches to distinct lineages of conspecific target DNA were found in two specific primers. Therefore, amplification failure may occur if the target thrips have high intraspecific genetic variation.

並列關鍵字

thrips ； molecular identification ； internal transcribed spacer ； specific primers

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防檢疫重要性薊馬（纓翅目：薊馬科）專一性引子之鑑定應用

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