紅檜暨臺灣扁柏個體鑑定分子標誌系統之建置

Abstract

紅檜與臺灣扁柏合稱檜木，是臺灣特有的本土植物，皆為臺灣中高海拔800-2900公尺針闊葉混交林帶的優勢樹種。檜木的樹型高大、材質極佳，為臺灣林業相當重要的珍貴資產。因檜木的價格高昂，造成非法盜伐事件層出不窮。林務及檢警人員即使能適時逮捕罪犯，但經常礙於證明贓木與犯罪現場的被害木是否為同一植株所需科學證據之不足，從而無法有效起訴罪犯。本論文以次世代定序(next generation sequencing, NGS)技術提供之序列資訊，篩選具多型性的簡單重複序列標誌(simple sequence repeat marker, SSR marker)，分別建立可用來鑑定紅檜與臺灣扁柏植株的個體鑑定系統，用來提供贓木和被害木比對的直接科學證據。 紅檜和臺灣扁柏皆同時使用DNA和RNA作為開發SSR標誌的序列來源。DNA和RNA的基因庫經Illumina平台定序後，紅檜和臺灣扁柏分別產生83,978,146及25,415,224的原始讀子。整理序列後紅檜和臺灣扁柏分別產生10,696,325和8,628,818的重疊群加單序列，分別篩選出383,918和381,543條含有SSR的序列。紅檜和臺灣扁柏分別設計了400組和500組SSR引子。紅檜以4個族群(北大武山、赫威神木、觀霧、向陽)共92個個體及臺灣扁柏以4個族群(太平山、思源啞口、大雪山、林道160)共96個個體進行標誌驗證。 紅檜一共開發了36個具有多型性的SSR標誌，對偶基因數在2至27間，觀察雜合度在0.000至0.891間，預期雜合度在0.103到0.906間，多型性信息含量在0.097至0.876間，有6個顯著連鎖群。以28個非連鎖的SSR標誌建立了紅檜的個體鑑定系統，累積相符率低至 1.652 × 10 -12，顯示即使紅檜族群數量達6,000萬株，仍然具有99.99%的信心水準，已超過紅檜的族群個體數(3206萬 ± 320萬株)。 臺灣扁柏一共開發了35個具有多型性的SSR標誌，對偶基因數在2至16間，觀察雜合度在0.000至0.802間，預期雜合度在0.041到0.872間，多型性信息含量介於0.058至0.858間，有4個顯著連鎖群。以30個非連鎖的SSR標誌建立了臺灣扁柏的個體鑑定系統，累積相符率低至 5.596 × 10 -12，顯示即使臺灣扁柏族群數量達1800萬株，仍然具有99.99%的信心水準，已超過臺灣扁柏的族群個體數(739萬 ± 73萬株)。 紅檜所開發的SSR標誌被用來進行紅檜樣品出處模擬分析，樣品回到正確出處的整體正確率為88.04%。未來在查扣贓木時，大致可以以基因型別初步判別紅檜贓木是來自臺灣島東部、西北或是西南部，可適當縮小需比對的被害木範圍。由於西部紅檜族群間有基因交流的情況，無法達到100%的正確率，因此，當贓木可能來自臺灣島西部時，仍需擴大檢視地理位置相近的族群。 而在臺灣扁柏的個體鑑定系統方面，以羅東林管處被盜伐的植株進行個體鑑定系統驗證，最終成功的將三件查扣贓木比對回被害木，這是臺灣第一件由分生技術證實贓木和被害木相符的案例，完成最初本論文所設定目標。此外，由盜伐案中也證實臺灣扁柏植株具有嵌合體現象，未來在以SSR系統進行贓木和被害木比對時，仍應謹慎與其他輔助證據合併使用。 紅檜暨臺灣扁柏個體鑑定分子標誌系統的建立，不僅可用來提供法院連結贓木和被害木的直接證據，本論文所開發的SSR標誌也是研究檜木族群遺傳及育種的重要參考資訊。

Chamaecyparis formosensis Matsum. and Chamaecyparis obtusa Sieb. & Zucc. var. formosana (Hayata) Rehder are Taiwan endemic cypress species and the dominant tree in the middle and high altitude (800-2,900 meters) mixed coniferous and broad-leaved forest in Taiwan. The cypress trees are tall with excellent wood quality, rendering them a precious asset to Taiwan's forest industry. Due to the high-priced timber of cypress, illegal logging incidents emerge in endlessly. Even though law enforcement authorities can arrest criminals in time, the scientific evidence that proves the stolen wood and the victim trees at the crime scene of the same individual is often unavailable. Thus, the criminals usually cannot be effectively prosecuted. In this thesis, the sequence information provided by next generation sequencing (NGS) technology was used to establish polymorphic simple sequence repeat (SSR) markers. Individual identification systems that can be used to identify C. formosensis and C. obtusa var. formosana for providing direct scientific evidence of stolen wood and victim trees. Both DNA and RNA were used as sequence sources for mining SSR markers of C. formosensis and C. obtusa var. formosana. After establishment of the DNA and RNA libraries by Illumina platform, 83,978,146 and 25,415,224 raw reads were generated from C. formosensis and C. obtusa var. formosana, respectively. After sorting the sequences, 10,696,325 and 8,628,818 contigs and singletons for C. formosensis and C. obtusa var. formosana were generated, respectively. Sequences containing SSRs were mined, yielding 383,918 and 381,543 sequences containing SSRs, respectively. Then, 400 and 500 SSR primer pairs were designed, respectively. C. formosensis was divided into 4 populations MM, HV, GW, and SY, a total of 92 individuals, whereas C. obtusa var. formosana was divided into 4 populations TP, QS, DS, and FR, a total of 96 individuals were used for marker verification. A total of 36 polymorphic SSR markers were developed in C. formosensis. The number of alleles for each markers is 2 and 27, observed heterozygosity and expected heterozygosity are ranged from 0.000 to 0.891 and 0.103 to 0.906. The levels of polymorphism information contentrange from 0.097 to 0.879. Six significant linkages were detected. The C. formosensis individual identification system was established with 28 non-linked SSR markers, and the combined probability of identity was as low as 1.652 × 10-12, which shows that even if the number of C. formosensis populations reached 60 million, greater than the known population size of 32.06 ± 3.2 million, it still has a confidence level of 99.99%. On the other hand, a total of 35 polymorphic SSR markers were developed in C. obtusa var. formosana. The number of alleles for each markers is 2 and 16, observed heterozygosity and expected heterozygosity are ranged from 0.000 to 0.802 and 0.041 to 0.872. The levels of polymorphism information contentrange from 0.058 to 0.858. Four significant linkages were detected. The C. obtusa var. formosana individual identification system was established with 30 non-linked SSR markers, and the combined probability of identity was as low as 5.596 × 10-12, which shows that even if the number of C. obtusa var. formosana populations reached 18 million, greater than the known population size of 7.39 ± 0.73 million, it still has a confidence level of 99.99%. The SSR mark developed by the C. formosensis was used to monitor the origin of the C. formosensis samples, and the overall correct rate of the samples returned to the correct source was 88.04%. In the future, when woods are seized, it may be possible to preliminarily determine whether the source of the C. formosensis wood is from the east, northwest, or southwest of Taiwan Island based on genotypes, which properly narrows the scope of the victim trees that need to be compared. However, due to the genetic exchange between the western C. formosensis groups, it is impossible to achieve a 100% accuracy rate. When the source of the wood may come from the western part of Taiwan Island, it is still necessary to expand the inspection of populations with similar geographical locations. The C. obtusa var. formosana individual identification was verified with the seized woods samples from Forestry Bureau Council of Agriculture of Executive Yuan, Taiwan, and successfully compared the three pieces of seized stolen wood back to the victim trees. This is the first case in Taiwan in which the molecular biology evidence was provided to prove that the stolen woods match with the victim trees scientifically, fulfilling the original goal set in this thesis. In addition, this case also confirmed chimeras in C. obtusa var. formosana. In the future, when using the SSR system to compare stolen wood and victim tree, it should still be used with caution in combination with other auxiliary evidence. The molecular marker system establishment of C. formosensis and C. obtusa var. formosana for individual identification not only provided direct evidence for the court to connect the relationship between the stolen wood and the victim trees but also provide essential reference information for studying genetics and breeding on cypress.