雙重酶切DNA定序技術應用於番椒種原基因體分析

次世代定序技術已成為作物基因體研究相當常見的工具，而為集中定序資源與降低定序成本，已逐漸發展出多種簡化基因體定序技術，然而不同作物、不同族群組成所適宜的文庫建置方法（如限制酶選擇或是定序量等）有差異，若能針對目標作物及族群組成進行方法上的調整，將可使定序資源發揮最大效率。本研究使用簡化基因體技術之一：雙重酶切DNA定序技術（double-digest restriction site-associated DNA sequencing; ddRAD-seq）針對不同種番椒（Capsicum spp.）種原基因體進行分析，第一部分比較PstI/MspI及PstI/MseI限制酶酵素組合，電腦模擬結果顯示兩種組合產生的可被定序片段為總片段之2.01%及0.34%，而實際2個酵素組合的定序結果顯示，以PstI/MspI組合（19-28M）比PstI/MseI組合（15-17M）得到較多成對且比對專一的讀序，因此不論在建庫資源利用上或是成功對應回參考序列的讀序數量／比例上，PstI/MspI皆較PstI/MseI表現佳。本研究第二部分針對28個番椒收集系（涵蓋5個番椒馴化種）以PstI/MspI酶切並進行ddRAD定序，共得到14,230個高品質單一核苷酸多型性（single nucleotide polymorphism; SNP），平均分布於番椒12對染色體並以染色體兩端基因較密集處較多；利用此些SNP進行集群分析，亦能成功將不同種番椒品系正確分類。此說明本研究使用的一系列建庫、定序、生物資訊分析等方法，可用於橫跨不同種的番椒基因體分析，未來將以此為基礎，針對台灣番椒種原建立其基因體資訊，作為台灣番椒育種的根基。

關鍵字

簡化基因體定序；雙重限制酶；番椒；基因體分析

並列摘要

Next-generation sequencing technologies lead to a new era of crop genetic research. To lower sequencing costs for building a single nucleotide polymorphism (SNP) map of a genome, the reduced representation genome sequencing technology was used to sequence the flanking DNA sequences of selected restriction enzyme sites of DNA samples. However, appropriate parameters for constructing the reduced representation sequencing library, such as the choice of restriction enzyme and sequencing depth, differ in target crops and population composition. As a result, methodological adjustments toward target crops and population composition are vital to maximizing the efficiency of sequencing resources. In this study, we presented a tailored double-digest restriction site-associated DNA sequencing (ddRAD-seq) genotyping method for pepper germplasm. In the first part of this work, the performance of two restriction enzyme pairs- PstI/MspI and PstI/MseI were assessed, and results of in silico simulation showed 2.01% and 0.34% fragments of the total digested fragment were sequenced, respectively. According to sequencing results, more uniquely mapped and adequately paired reads (19-28 M) were observed in PstI/MspI pair than in PstI/MseI (15-17 M). Therefore, in terms of resource utilization in library preparation, the enzyme pair PstI/MspI performed better than PstI/MseI. In addition, the number or ratio of reads that came from PstI/MspI digestion was more efficiently mapped to the reference genome. As for the second part of this work, the SNP maps of 28 pepper accessions belonging to 5 domestic species were constructed using the ddRAD-seq genotyping technique with PstI/MspI restriction enzyme combination. A total of 14,230 high-quality SNP were obtained and distributed evenly on the 12 chromosomes of pepper. Furthermore, as expected, the 28 pepper accessions were clustered into proper phylogenetic groups based on the principle component analysis and the dendrogram analysis. In summary, the ddRAD-seq genotyping technique was tailored to construct the SNP maps of the pepper germplasm in this study.

並列關鍵字

Reduced genome sequencing ； Double-digest restriction enzyme ； Capsicum ； Genome analysis

主題瀏覽