柑橘屬植物親緣關係及分子演化之研究

Abstract

柑橘屬植物之發展歷史悠久，過去對其親緣關係研究多根據其外觀型態以及化學特性等進行分類，然而柑橘屬植物之雜交種繁多，且有多種芽條變異而產生之品系，若僅以傳統植物分類方法往往易對其來源或分類有所誤判。近十幾年來因為分子技術進步，以分子標誌對生物重新進行分類上的探討已儼然成為分類學上的重要方法，在植物分類方面最常被使用的標的為葉綠體 DNA，主要是因為葉綠體在大多數植物中皆是母系遺傳，且演化速度較核基因慢，利於遺傳變異的分析，然而柑橘葉綠體中最常被用於親緣關係研究的僅有 trnL-trnL-trnF 非轉譯區域，其他區域鮮少被使用於遺傳變異與親緣關係的研究，因此本研究使用多個柑橘屬植物的核基因以及葉綠體基因進行遺傳變異上的分析並比較其差異。其中葉綠體基因之分析主要著重於其親緣關係之探討，核基因部份則以兩個與柑橘的環境適應和特性表現相關之基因進行研究。 第一部份以柑橘屬植物葉綠體上的9個非轉譯區域進行親緣分析，結果發現過去常被使用的 trnL-trnL-trnF 非轉譯區雖然具有高度的單套基因型歧異度，但在甜橙、葡萄柚和柚類中皆具有相同的單套基因型，僅適用於區分寬皮柑和甜橙。此外，葉綠體 DNA 上不同區域之演化速度不同，因而適用於分析不同種類的柑橘使用，若要得到最佳的區分效果，需要同時使用多個區域進行分析。 第二部份為使用細胞質第一型小分子熱休克蛋白進行其遺傳變異的分析，結果發現於轉譯區域的核酸變異大多為transversion 和 transition，並無 indel 出現，因而其胺基酸序列之保守性相當高。此基因的3’-UTR 有一段 indel 存在，此變異可將柚類、葡萄柚類以及橘柚 (tangelos) 依其原產地和環境適應性的差異進行區分，但在寬皮柑、甜橙、檸檬和萊姆中並無相同之效果，其可能是因為後者之特性較受人類喜愛，且可製造多種的加工產品，所以經過人為大量的進行雜交和配種，生產了許多適合栽種於各種氣候下的品系，造成以 cytosolic classI sHSP 核酸序列不易分群的情形。 第三部份為使用檸檬苦素葡萄糖轉移酶基因進行其遺傳變異的分析。柑橘屬植物中的甜橙類、寬皮柑類和葡萄柚類之檸檬苦素葡萄糖轉移酶多有兩種型態，而柚類中的暹邏柚 (S1) 和沙田柚 (S2) 以及 citron 中的佛手柑 (B1) 僅具單種型態之檸檬苦素葡萄糖轉移酶基因。對 24種柑橘屬植物的檸檬苦素葡萄糖轉移酶基因進行序列分析後發現，柑橘屬植物中之檸檬苦素類化合物的苦味應該是由多個因素所造成，並非僅與檸檬苦素葡萄糖轉移酶基因有關。

The discovery and development of citrus genus has a long history, however, in most of the past studies, citrus classification was mainly based on appearance and chemical characteristics. Citrus hybrids and branch mutations have made us difficult to distinguish their origins and species using traditional methods. Following to the advancement of molecular technology, the molecular markers have become the most important methods at taxonomy in past decade. The cpDNA (chloroplast DNA) has become one of the most useful markers for classification due to its characters such as maternal inheritance and slow evolution, which suitable for genetic variation analysis. The region commonly used for phylogenetic relationship of cpDNA is trnL-trnL-trnF noncoding region. The other regions were less used in the researches of phylogenetic relationship and genetic variation. In this study, nuclear DNA and cpDNA were used for the analysis of genetic variation. The noncoding regions of cpDNA were used to study their phylogenetic relationship. The genetic variation of two nuclear genes related to the environment adatpation and the fruit characteristic after processing were also compared. In the first part, 9 noncoding regions of cpDNA were used for the analysis of phylogenetic relationship of citrus. It was found out that the trnL intron and trnL-trnF IGS which frequently analyzed in the past showed high haplotype diversity. Nevertheless, the sweet oranges, grapefruits and pummelos have the same haplotype in these regions. Accordingly, both trnL intron and trnL-trnF IGS could only used to distinguish mandarins and sweet oranges. To address different evolutional rates of different regions at cpDNA and accuracy for analysis, we further combined several other DNA sequences. Secondly, the cytosolic classI small heat shock protein (sHSP) was used to analyze citrus genetic variation. It was found that most of the nucleotide variations in the coding regions were transversion and transition, and there was no indel in these regions. Therefore, the amino acid sequences of cytosolic classI sHSP were rather conservative. There was an indel in the 3’-UTR (3’-untranlated region) of this gene. According to this variation, the pummelos, grapefruits and tangelos which originated from different regions and grow in different climates could be distinguished into different groups. Nevertheless, this finding was not similar in the groups of mandarins, sweet oranges, lemons and limes. Latter citrus species were rapidly worldwide distributed and genetically hybridized and adatped to different climate patterns. These situations made the DNA sequences of cytosolic classI sHSP in citrus not an optimum marker for phylogenetic analysis. The final part in this study was to analyze the genetic variation of LGTase (limonoid glucosyltransferase). Most of the citrus, such as sweet oranges, mandarins and grapefruit, have 2 types of LGTase gene. However, 2 pummelos (Siam and Shatianyu) and one citron (Bergamot) only have 1 type of LGTase gene. Comparing the DNA sequences of LGTase from 24 citrus, it was found that the bitterness of limonoids was caused by several other factors instead of LGTase alone, thus its variation did not contribute much on limonoid bitterness.