蝴蝶蘭 (Phalaenopsis spp., 2N=38) 為蘭科多年生開花植物，是臺灣主要出口、外銷之花卉，在臺灣花卉產業之經濟產值佔有一席之地。姬蝴蝶蘭 (P. equestris) 為臺灣原生種蘭花，於2015年已完成全基因體解序。然而目前仍未有已發表之蝴蝶蘭遺傳圖譜。因此，本研究目的為建構蝴蝶蘭之遺傳圖譜。P. aphrodite subsp. formosana及 P. equestris為台灣唯二的原生種，也是蝴蝶蘭育種之重要親本。利用此二原生種雜交產生之118株第一子代，作為此遺傳圖譜之定位族群。本系吳文鑾教授長期專注於開發簡單重複序列 (simple sequence repeat, SSR) 分子標誌，包含利用表現序列標籤(expressed sequence tags, ESTs)，454定序DNA序列，bacterial artificial chromosome (BAC)-end sequences (BESs) 等共發展蝴蝶蘭5,483 SSRs，且其中603 SSRs已設計引子序列。本研究利用其中333 SSRs於父母本間進行多型性 (polymorphism) 之篩選，其中241 (72.37%) SSRs可成功於6% polyacrylamide gel electrophoresis (PAGE)分析產生增幅條帶，而162 (48.65%) SSRs於父母本間顯示出多型性。此外，先前實驗室以genotyping-by-sequencing (GBS) 技術發展了2,768單一核苷酸多型性分子標誌(single nucleotide polymorphisms, SNPs)。以卡方適合度檢定 (chi-square test) 篩選SSRs及SNPs分子標誌，其中108 SSRs和1,798 SNPs共1,906分子標誌可被應用於此遺傳圖譜之建立。利用OneMap軟體進行連鎖分析 (linkage analysis)，最終總共1,214分子標誌成功定位於27連鎖群 (linkage groups, LGs) 構成之連鎖遺傳圖譜。此圖譜總長約15,192 centimorgan (cM)，每個分子標誌間平均距離為12.51 cM。和P. equestris scaffold序列以及BESs 比對後，將27 LGs 分群成可能之19 homologous groups (HGs)。本遺傳圖譜提供未來進行數量性狀基因座 (quantitative trait loci, QTL) 定位以及進一步分子輔助育種 (marker-assisted selection, MAS) 之基礎。

Phalaenopsis orchids (2N=38) are perennial flowering plants, hold a significant economic value in Taiwan agricultural industry, and are the major export of ornamental crops in Taiwan. Whole genome sequencing of P. equestris, a native species in Taiwan, has been completed in 2015. However, the genetic linkage map for Phalaenopsis is still unavailable. The aim of this thesis is to construct a genetic linkage map for Phalaenopsis. Both P. aphrodite and P. equestris are the only two native Phalaenopsis orchids in Taiwan. For constructing the genetic map, a F1 population consisting of 118 progenies from a cross between P. aphrodite and P. equestris was used. Dr. Wen-Luan Wu has being for long focused on developing simple sequence repeat (SSR) of Phalaenopsis orchids. Her group had developed 5,483 SSRs from expressed sequence tags (ESTs), 454 sequenced genomic DNA and bacterial artificial chromosome (BAC)-end sequences (BESs). Among them, 603 SSRs have primer sequences designed. In this study, 333 SSRs with primer sequences were used. 241 (72.37%) SSRs can be successfully amplified, and 162 (48.65%) SSRs revealed polymorphism between two parents by using a 6% polyacrylamide gel electrophoresis (PAGE). In addition, 2,768 single nucleotide polymorphism (SNP) markers have been previously developed by using genotyping-by-sequencing (GBS) analysis in our lab. SSR and SNP markers were filtered through chi-square test and eventually 108 SSRs and 1,798 SNPs, total 1,906 markers were applied for linkage analysis. The genetic linkage map was constructed by OneMap software, a package from R software. It is comprised of 27 linkage groups (LGs) with total 1,214 mapped markers. The total length of 27 LGs is 15,192 centimorgan (cM), and the average marker density is 12.51 cM. The 27 LGs then were assembled into potential 19 homologous groups (HGs) according to scaffold sequences that have already paralleled with BESs. The genetic linkage map of Phalaenopsis will facilitate the access of genes that correspond to the interested traits and quantitative trait loci (QTL), incorporate with marker-assisted breeding, and enhance genome assembly of P. equestris.

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