開發分子標誌以鑑定牛樟、樟樹、冇樟與種間雜交評估

牛樟(Cinnamomum kanehirae Hayata)為臺灣特有林木物種，也是重要貴重樹種，近年來，由於天然寄生於牛樟木的牛樟芝價格不斐，更提高牛樟的使用需求，進而使得牛樟生產、鑑定、溯源甚至是保育遺傳的相關研究也孕育而生。本研究希望透過DNA分子標誌，在牛樟分類上進行有效的鑑定工作。本研究收集牛樟樣本，以次世代定序技術，建立牛樟轉錄體基因組序列，組裝分析後得到58,950個unigene，並從中初步驗證94組微衛星體序列(Microsatellite, Simple sequence repeat，SSR)，從中挑選使用11組SSR進行牛樟、冇樟(C. micranthum)、樟樹(C. camphora)的種間遺傳分析，可以明顯從主座標分析中看出三個物種的分群，但是卻無法進行牛樟、樟樹、冇樟間的快速分子鑑定。為了達成牛樟、樟樹、冇樟種間的快速鑑定，以次世代定序技術，得到牛樟、樟樹、冇樟的全葉綠體基因組序列，藉由全葉綠體基因組間的序列變異，開發插入缺失序列(insertion/deletion，InDel)作為鑑定有分類爭議的牛樟與冇樟，提出可快速鑑定牛樟與冇樟樣本的6組InDel序列，以及對牛樟與冇樟葉片甲醇萃取物進行成分分析，其中牛樟葉片甲醇萃取物中含有芳樟醇與芝麻素，但冇樟卻無此成分，也可做為鑑別2者差異的化學分子標誌。然而，牛樟與樟樹的全葉綠體基因組序列分析中，葉綠體InDel序列無法將牛樟與樟樹進行區分鑑定，主要的原因在於牛樟與臺灣東部樟樹葉綠體序列相似，在親緣關係圖上也表示出牛樟與東部樟樹遺傳距離較近，而與西部樟樹族群的葉綠體序列有較大的序列變異，在葉綠體InDel序列上，牛樟與東部樟樹族群樣本屬於同樣的基因型，因此無法進行有效鑑定。因此，本研究進一步透過GATK(Genome Analysis Tool Kit )流程進行牛樟、樟樹、冇樟全基因組低覆蓋度定序分析，並相互比對組裝的基因資料庫，開發種間全基因組的InDel序列，有5組InDel可以成功用於牛樟與樟樹種間的快速鑑定，但也進一步發現無法透過InDel序列鑑定區別牛樟與雜交牛樟樣本。為了深入解決牛樟與雜交牛樟鑑定與評估，進一步藉由簡化基因組定序技術(Reduced representation libraries)，得到牛樟、樟樹與雜交牛樟的全基因組中差異的SNP位點，最終分析840S個SNPs，再搭配除具有母系遺傳與共顯性特性的InDel序列，確定雜交的母本為牛樟，證實牛樟、樟樹間出現天然的雜交牛樟個體，透過遺傳結構分析，表示牛樟與樟樹的雜交是屬於雙向的，並且已經產生F1、F2與回交的後代，雜交發生的原始區域推測應為臺灣東部，且多發生於人為開發區域的種子園、苗圃或是行道樹。因此，在尚未完全了解雜交牛樟特性，以及雜交是否對於純牛樟或是純樟樹的基因庫產生汙染，未來在生產牛樟種子或是苗木時，在牛樟種子園周遭應該設立緩衝帶，避免樟樹跨樹種間的雜交，確保牛樟基因庫的維持與保育，同時，SNP的分析結果也發現分布於臺灣東部西南部與西北部的樟樹族群間，存在分子結構的差異。本研究透過SSR、葉綠體InDel、基因體InDel與SNP，針對牛樟、樟樹、冇樟與雜交牛樟進行種間分子鑑定，有助於正確培育牛樟苗木，甚至有效保存復育牛樟族群，未來牛樟復育策略，應該配合分子技術，將可以有效正確鑑定牛樟，甚至分析牛樟種源與遺傳特性，有助於牛樟資源的復育與育種工作。

關鍵字

牛樟；分子標誌；插入缺失序列；微衛星體序列；單一核苷酸多型性；樹種鑑定；雜交鑑定

並列摘要

Cinnamomum kanehirae Hayata is an endemic and important precious tree species in Taiwan. In recent years, due to the high price of Antrodia cinnamomea which is naturally parasitized on C. kanehirae, the demand of C. kanehirae are increased. Thus, the relevant researches are necessary, such as species identification, origin traceability and genetic conservation. This study focuses on species identification of C. kanehirae by using molecular markers. In the results of C. kanehirae transcriptome sequencing, 58,950 unigenes were obtained after de novo assembly, and 94 SSR molecular markers were developed and preliminarily verified. Evelen SSRs were used to conduct interspecies genetic analysis of C. kanehirae, C. camphora, and C. micranthum. The PcoA shows the clearly three separate groups in C. kanehirae, C. camphora, and C. micranthum. It shows the genetically diefference between these three species. However, the analysis of SSR markers is not a rapid method for species identification. In order to develop a rapid identification method, the comparative analysis of complete chloroplast genomes can help develop useful DNA markers for species identification. We provided six chloroplastic InDels to solve the controversially taxmomic event between C. kanehirae and C. micranthum. High amounts of linalool and sesamin were present in the methanol extracts of C. kanehirae leaves, but not in C. micranthum leaves. These chemical profiles of methanol extraction of C. kanehirae and C. micranthum leaves are also could be used for chemical markers. Unfortunately, the chloroplast InDel can not be use for distingulishing C. kanehirae and C. camphora. The chloroplast genomes of C. kanehirae and easrern and south wastern C. kanehirae are too similar with each other to design suitable Indel regions. In the phylogenetic analysis which was based on Luraceae complete choloplast genomic sequences shows that the C. kanehirae is closer to C. camphora (JLH2) than to C. micranthum and northern and western C. camphora. Because the chloroplst InDel could not for identification effectively, the genomic InDel markers were developed by using low-coverage sequencing with GATK (Genome analysis tool kit) pipeline. These InDel markers can be used for rapidly species identification. Five InDels were successfully used for identification between C. kanehirae and C. camphora. However, the putative hybrids are differently to be distingulished from C. kanehirae. Due to doubts about the hybridization between C. kanehirae and C. camphora for many years and the objective of successful hybrids identification. The reduced representation library was used for developing single nucleotide polymorphism (SNP). Eventually, 840 SNPs and maternal inheritance, codominant InDel sequences were used for hybridization analysis. The results of 840 SNP genetic analysis indicate that the naturally hybridization happened between C. kanehirae ans C. camphora. Hybridization originally occurred in the human-mediated areas of eastern Taiwan and the introgression was bidirectional. Hybrids even could be classified as F1 (C. kanehirae x C. camphora), F2 and backcrosses. For producing pure wood or conservation aspects, the buffering-zones should be established for the seed orchard to avoid the cross-species pollination and to preserve the pure genetic composition of C. kanehirae. Results of SNP analysis also found that there are genetic differences between the C. camphora populations in eastern and western regions of Taiwan. In present study, SSR, chloroplastic and genomic InDel, and SNP were successful for identification of C. kanehirae, C. micranthum, C. camphora and hybrids. In order to ensure the correct cultivation of C. kanehirae, and even to effectively preserve the high genetic diversity of C. kanehirae, more active restoration strategies should be adopted in the furture. Therefore, the DNA markers provided in this study and molecular genetic technologies sholud be applied for C. kanehirae genetic analysis such as species identification, provance and also could be contributed to the future restoration and molecular breeding of C. kanehirae.

並列關鍵字

Cinnamomum kanehirae ； molecular marker ； insertion/deletion (InDel) ； microsatellite (SSR) ； single nucleotide polymorphism (SNP) ； species identification ； hybridization identification