苦丁茶冬青及其近缘种的研究与种质资源评价
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摘要
“苦丁茶”是中国民间一大类代茶饮料植物的总称。其中,有两类”苦丁茶”在国内外比较有影响：一类是以苦丁茶冬青Ilex kudingcha为代表的冬青科苦丁茶；另一类是以粗壮女贞Ligustrum robustum为代表的木犀科苦丁茶。本研究仅限于研究冬青科苦丁茶。冬青科苦丁茶包含苦丁茶冬青I. kudingcha、大叶冬青I.latifolia、五棱苦丁茶I.pentagona、枸骨I. cornuta、华中枸骨I. centrochinensis、霍山冬青I. huoshanensis等136种冬青属植物。在6种植物当中,栽培面积、产量和经济价值等方面最具影响者系苦丁茶冬青。影响冬青属苦丁茶植物产业发展和推广的因素较多,其中植物基源和种质资源品质是两大关键因素。由于传统上冬青属植物归属于分类较为困难的群体之一,在植物系统学上表现为分类学者对是否应将苦丁茶冬青与扣树I.kaushue,以及霍山冬青与华中枸骨给予合并存在着争议。此外,海南大学苦丁茶研究所还发现了存在系统位置不明的冬青属苦丁茶种质材料,其形态特征介于苦丁茶冬青和五棱苦丁茶之间。这些系统学难题给冬青科苦丁茶的可持续发展带来了严重的负面影响。本研究从植物形态学、解剖学、微形态学、核型特征、数量分类学、化学分类学、红外光谱分析、分子生物学等角度对苦丁茶冬青及其近缘种的系统学进行比较系统的和综合研究,以期为解决冬青属苦丁茶系统学的难题提供科学依据。此外,作者还以绿原酸和芦丁为标准,利用反相高效液相色谱和超声提取等方法对苦丁茶冬青及其近缘种的种质资源进行科学评价,为其资源开发和利用提供理论支撑。本研究取得的主要成果如下：
1.对6种冬青属苦丁茶植物形态进行了观察和比较,结果显示,本研究的物种茎的形态有五棱形和圆形两类。苦丁茶冬青的花被基数、子房室数目、果实分核数目和花的性别特征均存在丰富的多样性。上述特征对于冬青属苦丁茶植物的分类鉴定和遗传多样性的研究具有一定的辅助意义。
通过实地观测,结果表明,除五棱苦丁茶的植物茎为五棱形外,其余5种植物的茎均为圆形。苦丁茶冬青的花被数、子房室数目、果实分核数目以4为主；在性别方面,以单性花为主,但也存在杂性的现象。
2.对6种冬青属苦丁茶植物茎和叶柄的解剖结构进行了观察和比较。结果显示,本研究的物种之间其茎的结构大体相似,其差异体现在髓部形态、髓部与次生维管束宽度比例、木射线的宽度及密度方面。该6种冬青属植物叶柄解剖结构也大体相似,其主要差异体现在维管束的形态方面,可分为两大类。
利用荧光显微镜观察了茎和叶柄的解剖结构。结果表明,霍山冬青茎中髓部宽度所占比例最大；枸骨和华中枸骨茎的髓部形态呈四方形,其他四种茎的髓部形态呈圆形或近圆形；木射线最宽的是苦丁茶冬青,大叶冬青和五棱苦丁茶的木射线宽度次之；叶柄木质部束呈较规则半月形者为枸骨、华中枸骨、霍山冬青和大叶冬青,典型不规则半月形者为苦丁茶冬青和五棱苦丁茶。
3.6种冬青属苦丁茶植物的茎叶形态数量分类学研究结果与叶柄解剖分类结果相似,均可分为同样的两大类。
基于26个茎叶形态变量的数量聚类分析结果,可将6种冬青属苦丁茶植物分为两大类,第一类为苦丁茶冬青和五棱苦丁茶；第二类为大叶冬青、霍山冬青、枸骨和华中枸骨4种植物。主成分分析结果表明第一主成分主要反映的是叶片对称性、叶柄长度、叶基到最宽处的距离与叶长的比值。
4.叶表皮微形态特征研究结果表明,冬青属苦丁茶叶片微形态特征具有种级水平上的分类学意义,可以为苦丁茶冬青及其近缘种的划分提供可靠的依据。根据叶表皮微形态特征的研究结果,不支持霍山冬青与华中枸骨的合并。结合分子生物学证据与叶表皮特征微形态特征的研究结果,形态居间种质材料的冬青属苦丁茶类型应为五棱苦丁茶(Lpentagona)的变种。
光学显微镜和扫描电镜研究结果表明,7种冬青属苦丁茶植物的叶表皮微形态特征具有一定的相似性,如气孔器均位于下表皮,每种植物均有正常气孔器和大气孔器,气孔器类型以环列型为主,其次为双环列型；其差异性体现在叶表皮细胞的垂周壁形态和气孔外缘角质层纹饰等方面。霍山冬青叶表皮微形态与华中枸骨存在着较大区别。
5.观察了不同地域的枸骨和四种性别特征材料的苦丁茶冬青的正常与异常花药与花粉的微形态学特征,同时还研究了大叶冬青、贡山冬青和猫儿刺的花粉特征。研究结果表明,花药纹饰大体相似,但花粉外壁纹饰和萌发沟、花粉体积在种间和种内具有较大差异。不育或败育花粉则在萌发沟深度和长度方面与正常花粉具有较大差异。根据花药与花粉微形态学研究结果,说明花药特征对冬青属系统分类意义不大,但花粉的特征对于冬青属植物的分类具有一定的价值。
扫描电镜研究结果表明,所研究的五类材料其花药纹饰大体相似,都具有不规则脊纹并间有条纹,种间区别不显著；花粉均为三沟型,外壁纹饰均具疣状或钉状颗粒。不同物种间、不同地域的枸骨与不同性别特征的苦丁茶冬青其花粉体积大小指数、纹饰颗粒密度、萌发沟长度和宽度及其比值等特征都具有多样性。此外不育或败育花粉微形态体现在萌发沟深浅和长度不一,具有一定的生态学意义。
6.染色体核型分析结果表明,4种冬青属苦丁茶的核型可分为两类,核型分析的研究结果有助于研究冬青属植物的亲缘关系。
通过酶解离染色后显微镜观察,发现苦丁茶冬青、大叶冬青和形态居间类型种质材料其核型属于2B类型,五棱苦丁茶属于3B类型。就核型分析结果而言,形态居间类型种质材料的核型更接近于苦丁茶冬青。
7.冬青属苦丁茶化学分类学聚类分析研究结果显示,五棱苦丁茶与苦丁茶冬青归为一类,这表明化学分类学与形态数量分类学研究结果基本一致；同时也显示,苦丁茶冬青在化学成分方面具有一定的多样性,这种多样性与其分布地域有关。
根据RP-HPLC数据和NTsys软件进行了化学分类学研究。六种冬青属植物中色谱峰共有峰数目有6个,另外在各个物种色谱图中,作者还发现了一些特殊峰。根据聚类分析的结果,五棱苦丁茶(S12,S13)与苦丁茶冬青(S2)种质材料聚类在一起,其亲缘关系最近,但起源于广西马山石灰岩地区的苦丁茶冬青(S1)种质材料单独聚为1类,其所含有的化学成分种类及含量与其他地域苦丁茶冬青的差异很大。
8.六种冬青属苦丁茶的叶片因种质材料的起源地域,野生来源与栽培条件以及叶片储藏措施的不同,其绿原酸含量存在明显差异。
基于超声提取和RP-HPLC基础上的绿原酸含量研究结果表明,新采集的成熟功能叶经过低温干燥后,其所含绿原酸含量由大到小依次为丁茶冬青>霍山冬青>大叶冬青>五棱苦丁茶>华中枸骨>枸骨；干燥的成熟功能叶放置2月后绿原酸含量显著降低；在苦丁茶冬青野生材料不同发育阶段的叶片中,大部分材料以老叶绿原酸含量最高；一般来说,苦丁茶冬青野生材料叶片中绿原酸含量要高于栽培材料,种间或种内绿原酸含量存在较大的差异,这可能与种质材料的起源地域有关。
9.根据叶总黄酮含量测定结果,并结合绿原酸含量的分析结果,可以认为霍山冬青是一种良好的苦丁茶种质资源；利用幼嫩叶加工成苦丁茶具有其科学性。
在超声提取、络合反应及紫外分光光度法,以芦丁为标准的含量测定结果表明,就5批次栽培材料的功能叶总黄酮的平均值而言,苦丁茶冬青(3.96%)>五棱苦丁茶(3.67%)>霍山冬青(3.63%)>枸骨(3.04%)>大叶冬青(2.64%)；低温干燥(50℃)放置2月后的叶片总黄酮含量明显降低；栽培条件下的苦丁茶冬青,其成熟叶片中的总黄酮含量平均值要高于野生植株成熟功能叶所含量；就所有个体总黄酮含量而言,霍山冬青最高(7.39%)；幼嫩叶的总黄酮含量具有较高的水平。
10.基于红外光谱分析的研究表明,利用红外二阶导数谱图可以将不同物种的冬青属苦丁茶植物和不同来源的苦丁茶冬青种质材料快速和有效地加以区分。
一维傅里叶中红外光谱分析表明,不同物种的冬青属苦丁茶植物和不同来源的苦丁茶冬青种质材料不易区分,有大量的共同的吸收峰,特征峰相对较少。通过对指纹区二阶导数图谱变换,将重叠的光谱分开后,除共同的吸收峰外,不同物种的冬青属苦丁茶植物和不同来源的苦丁茶冬青种质材料叶片红外光谱图吸收峰位置、数目和吸光度显示出了明显差异,利用此差异可以对上述物种或不同来源的种质材料进行鉴别。利用红外光谱聚类分析表明,形态居间类型种质材料与五棱苦丁茶亲缘关系较近,霍山冬青与华中枸骨亲缘关系相对较远。
11.基于SRAP-PCR分子标记的聚类分析结果表明,供试的6种冬青属苦丁茶可分为两大类,与其他分子标记的实验结果基本一致。同时SRAP分子标记实验结果还显示供试的苦丁茶冬青不同的种质材料可分为两大类,这己研究结果为探讨苦丁茶冬青的基因型分类提供了可靠的科学证据。
在本研究中,苦丁茶冬青、五棱苦丁茶和大叶冬青聚为一大类,枸骨、华中枸骨和霍山冬青聚为一大类,这与传统的根据其叶形的分类结果完全一致。也与其他分子标记实验的结果相吻合。因此,SRAP分子标记用于冬青属苦丁茶植物分子系统学研究以及种级水平和种下水平分类鉴定是完全可行的。另一方面,根据聚类分析结果,不同来源的供试苦丁茶冬青种质材料还可分为2个类群,每个类群又可分为若干小类。这说明苦丁茶冬青存在比较丰富的遗传多样性。
本论文在综合分析各种研究手段的实验结果基础上,对冬青属苦丁茶植物的系统亲缘关系及优良种质资源选择进行了探讨。
"Kudingcha" was the general name for a large class of Chinese folk tea beverage plants. There were two types of "Kudingcha" with a large impact at home and abroad among of them. One type of "Kudingcha" was made of species from Aquifoliaceae with the representative species of Ilex kudingcha. The other type of "Kudingcha" was made of species from Oleaceae with the representative species of Ligustrum robustum. This study was limited to the species of Ilex L.(Aquifoliaceae) used as "Kudingcha". The Aquifoliaceae origins of Kudingcha were I. kudingcha and its related species ncluding I.latifolia, I.pentagona, I.huoshanensis,cornuta and I.centrochinensis. Among of six species,the largest of the planting area,yield and economic value was I. kudingcha. There were many factors influencing the industry development and promotion of Kudingcha from Ilex species, and the two key factors were their botanical origin and quality of germplasm resource. But Ilex L.was a groups with more difficult classification, and their had a controversial among different scholars on whether there should merge I. kudingcha and I.kaushue; I.huoshanensis and I.centro-chinensis. In addition, intermediate species with indecisive systematic position (I.sp.) have been revealed by Kudingcha Research Institute of Hainan University, and ts morphological characteristics was intermediated between I. kudingcha and I.pentagona. It have brought a serious influence on sustainable development for Kudingcha. In order to provide scientific basis for solving systematic problems, we have studied the systematic of I. kudingcha and its related species from the perspective of plant morphology, anatomy, micro morphology, numerical taxonomy, chemotaxonomy, infrared spectral analysis, molecular biology. And in order to provide theoretical support for the development and utilization of germplasm resources, Author also have evaluated the germplasm of I. kudingcha and its related species by using methods of RP-HPLC, ultrasonic extraction with standard of chlorogenic acid and rutin. The main results were as follows.
1. Author have observed plant morphology from six Ilex species used as kudingcha. The results showed that stem form of the studied species had five prismatic and circular form. There were abundant diversity for I. Kudingcha in gender and number of fruit, perianth,and locules nuclear of fruit. The above characteristics could have auxiliary meaningful for classification and genetic diversity for Ilex species used as Kudingcha.
Through field observation, author have found that, stem form of I.pentagona was five prismatic and other five species were circular form. The main number were four in number of fruit, perianth and locules nuclear of fruit. In terms of gender, the main gender were unisexual flowers, but there were polygamous phenomenon.
2. Author have observed the anatomy structure of stem and petiole from six Ilex species used as Kudingcha. The results showed that stem structure similar to each other.The differences were the width and density of wood ray, medullary morphology, and the ratio of pith and secondary bundle width.Petiole anatomical structures of the six aboved species are similar to each other too.The difference lies in the form of vascular bundle, and they can be divided into two categories.
Author found that the largest proportion of stem pith width among six species was I.huoshanensis according to the anatomical structure of stem and petiole under the fluorescence microscope. Medullary morphology of stem from I.cornuta, I.centrochinensis was four square, and the other four species were round or nearly round; The species with most wide in wood ray was I.kudingcha, The second was I.latifolia and I.pentagona; The species with regular half moon petiole xylem are I.cornuta, I.centrochinensis I.huoshanensis, and I.latifolia.irregular one are I.pentagona and I. kudingcha.
3.Results of research of stem and leaf morphological quantitative taxonomy of six Ilex species showed that there have similar to the results of petiole anatomy classification, and they could be divided into the same two categories.
The reults of cluster analysis of six Ilex species used as Kudingcha could be divided into two categories based on26morphological variable from stem and leaf.The first class were I.kudingcha and I.pentagona, second for I.latifolia, I.huoshanensis, I.cornuta, I.Centrochinensis.incipal component analysis (PCA) showed that the first principal component mainly reflects the blade symmetry, petiole length, the ratio of distance of base to largest leaf width and leaf length.
4.The results proved that the micro-morphological characteristics of leaf epidermis of Ilex L. had taxonomic significance at the species level, it could be used as reliable evidence in distinguishing I.kudingcha with its related species.There should not be treated I.huoshanensis and I.centrochinensis as the same species according to their micro-morphological characteristics of leaf epdermis.Combined with the evidence of molecular biology and the characteristics of the leaf epidermis, intermediate types should be treated as varieties of I.pentagona.
Micro-morphological characteristics of leaf epdermis for I. kudingcha and its six relative species were examined with an optical microscope and SEM. Micro-morphological structure of leaf epidermis for seven Ilex species as Kudingcha had a certain similarity such as the stomata at lower epidermis.According to the size of stomata, it could be divided into two typies of stomata:large stomata and common stomata.Most of stomatal type were cyclocytic.There also had some differences in leaf epidermis characteristics, such as leaf epidermal cells and stomata anticlinal walls form, the outer edge of the cuticle ornamentation of stomata, etc. And there have a big difference in leaf epidermal morphology between I.huoshanensis and I.centrochinensis.
5. The micro-morphological characteristics of different regions of I.cornuta and four gender characteristics of materials of normal and abnormal anther and pollen of I.kudingcha have been observed. At the same time, the pollen characteristics of I. latifolia,I. hookeri and I. pernyi have also been studied.The results showed that, the anther ornamentation of studied materials were similar to each other. But there had a great difference in the pollen exine sculpture, germinal furrow and pollen size among interspecific and intraspecific level.There had a larger difference between infertility or sterility and normal pollen in germinal furrow depth and length. According to the anther and pollen micro-morphological characteristics, anther characteristics was little significance in classification of Ilex species, but the pollen characteristics had a certain value for the classification of Ilex species.
The results under SEM was showed that, the anther ornamentation was similar to each other among of studied materials. The anther had irregular ridges and striped, cxine sculpture of pollen had verrucous particles and there was no significant differences in anther among Ilex species. Pollen had three germinal furrow. And the exine have ornamentation with verrucous or nail shaped particles. It was diverse in pollen size index, particle density, length and width and its ratio of germinal furrow characteristics among different species, different regions of I.cornuta and different gender characteristics of I. kudingcha. In addition, the characteristic of morphology of infertility or sterility pollen were irregular length and depth of germinal furrow, It could help to clarify some ecological significance.
6.The results of karyotype analysis showed that the four Ilex species could be divided into two classes, and it could help the study of phylogenetic relationships among Ilex species for karyotype analysis.
By using enzymatic dissociation and staining, the results showed that karyotype of I.kudingcha, I.latifolia and intermediate type(I.sp) belongs to2B type, I.pentagona belongs to the type of3B under the microscopy, and it also showed that the form of intermediate type(I.sp)close to that of I.kudingcha according to the feature of karyotype.
7. The results of cluster analysis based on chemotaxonomy showed that I.kudingcha and I.pentagona belonged to the same class, it showed that the chemotaxonomy and morphological quantitative taxonomy have the same results; At the same time it showed that I.kudingcha had a certain degree of diversity in chemical composition, and it may be related with its geographical distribution.
Author have found six common peaks according to chemical taxonomy research based on chromatographic data from RP-HPLC method and Ntsys software. In addition, we also have found some special peak from all chromatograms in six species.According to the results of clustering analysis, Ⅰ.pentagona,(S12, S13)and I.kudingcha(S2) clustered together, the two species had the closest relationship.But I.kudingcha (S1) which come from Guangxi Province, Mashan limestone areas have clustered into one categories alone.There have a big difference both in chemical components and contents combared with the other regions of the I.kudingcha, It may be related to that germplasm materials may have formed a chemical type.
8. Owing to different geographical origin of germplasm, wild and cultivation conditions, storage measures of leaves for six species of Ilex L., there were obvious differences in the content of chlorogenic acid.
The content of chlorogenic acid of fresh mature leaves of six species from high to low is I.kudingcha> I.huoshanesis> I.latifolia> I.centrochinensis> I.pentagona> I.cornuta which have been determined by ultrasonic extraction and RP-HPLC method.; and the content of fresh leaves was much larger than that of the dried up to two months; for content of chlorogenic acid of different developmental stages of wild I.kudingcha samples, old leaves with infected> mature leaves> young leaves.The mean content of chlorogenic acid in the mature leaves of the wild was larger than that of cultivation one for I.kudingcha.There was much difference among six Ilex species and intraspecific of I.kudingcha according to chlorogenic acid content which may be related to the origin of species.
9.The results showed that I.huoshanesis was a better germplasm resources combined with content determination of both the chlorogenic acid and rutin, and it had a scientific significance that people used the tender leave as materials of Kudingcha.
The content of total flavonoids (rutin) from5species of Ilex L.used as kudingcha tea were determined using ultrasonic extracti, complexation reaction and UV spectrophotometry.On the average of value of total flavonoids from5batches of planting materials, I.kudingcha (3.96%)> I.pentagona(3.67%)> I.huoshanesis (3.63%)> I.cornuta (3.04%)> I.latifolia (2.64%); and total flavonoids in the leaves of placed after two months at low temperature dried has decreased significantly; On the average, the content of total flavone content of cultivation of mature leaves of I.kudingcha was significantly higher than that of wild mature leaves.The content of total flavonoids of I.huoshanesis was highest (7.39%) in all individuals from five species; Content of total flavonoids in young leaves maintained a higher level.
10. The results of infrared spectrum analysis of germplasm materials from I.kudingcha and its relatived species based on the second derivative spectra showed that Ilex species used as Kudingcha and different sources of I. kudingcha could be quickly and effectively distinguished with each other.
The one-dimensional fourier infrared spectrum analysis showed that, germplasm materials from Ilex species used as kudingcha and different sources of I. kudingcha were not easy to distinguish and there were a lot of common peaks. The number of characteristic absorption peak was relatively small. The overlapped spectra could be separated by using second derivative spectrum transform of the fingerprint region, In addition to a common peak, it showed obvious differences in IR absorption peak position, number and absorbance among of different species and germplasm materials with different sources of I. kudingcha. Thus it could be identified to those species. The results of clustering analysis by using the infrared spectral have been showed that intermediate species (I.sp) was close to I.pentagona in phylogenetic relationship,and I.centrochinensis had a relatively distantly relationship to Lhuoshanensis.
11. The results of cluster analysis based on the SRAP molecular markers showed that six Ilex species used as Kudingcha could be divided into two categories, which were consistent with experimental results on the basis of leaf morphology and other molecular marker. At the same time, the research results showed that Lkudingcha could be divided into two categories, which provides reliable scientific evidence for the genotypic classification of Lkudingcha.
In this study, I.kudingcha, I.latifolia, I.pentagona had been clustered into one category, I.cornuta, I.centrochinensis, Lhuoshanensis have been clustered into another category, it was consistent with the traditional classification according to its leaf morphology and other molecular marker results. Therefore, it was feasible that SRAP molecular markers could be used as systematics research of identification In the level of species and subspecies.On the other hand, according to the results of clustering analysis, Lkudingcha also could be divided into2categories, and each category can be divided into several small groups.It showed that. I.kudingcha had greater genetic diversity.
Plant systematic and excellent germplasm resource selection among of I.kudingcha and its related species have been discussed on the basis of comprehensive analysis of the experimental results from various research methods.
1.对6种冬青属苦丁茶植物形态进行了观察和比较,结果显示,本研究的物种茎的形态有五棱形和圆形两类。苦丁茶冬青的花被基数、子房室数目、果实分核数目和花的性别特征均存在丰富的多样性。上述特征对于冬青属苦丁茶植物的分类鉴定和遗传多样性的研究具有一定的辅助意义。
通过实地观测,结果表明,除五棱苦丁茶的植物茎为五棱形外,其余5种植物的茎均为圆形。苦丁茶冬青的花被数、子房室数目、果实分核数目以4为主；在性别方面,以单性花为主,但也存在杂性的现象。
2.对6种冬青属苦丁茶植物茎和叶柄的解剖结构进行了观察和比较。结果显示,本研究的物种之间其茎的结构大体相似,其差异体现在髓部形态、髓部与次生维管束宽度比例、木射线的宽度及密度方面。该6种冬青属植物叶柄解剖结构也大体相似,其主要差异体现在维管束的形态方面,可分为两大类。
利用荧光显微镜观察了茎和叶柄的解剖结构。结果表明,霍山冬青茎中髓部宽度所占比例最大；枸骨和华中枸骨茎的髓部形态呈四方形,其他四种茎的髓部形态呈圆形或近圆形；木射线最宽的是苦丁茶冬青,大叶冬青和五棱苦丁茶的木射线宽度次之；叶柄木质部束呈较规则半月形者为枸骨、华中枸骨、霍山冬青和大叶冬青,典型不规则半月形者为苦丁茶冬青和五棱苦丁茶。
3.6种冬青属苦丁茶植物的茎叶形态数量分类学研究结果与叶柄解剖分类结果相似,均可分为同样的两大类。
基于26个茎叶形态变量的数量聚类分析结果,可将6种冬青属苦丁茶植物分为两大类,第一类为苦丁茶冬青和五棱苦丁茶；第二类为大叶冬青、霍山冬青、枸骨和华中枸骨4种植物。主成分分析结果表明第一主成分主要反映的是叶片对称性、叶柄长度、叶基到最宽处的距离与叶长的比值。
4.叶表皮微形态特征研究结果表明,冬青属苦丁茶叶片微形态特征具有种级水平上的分类学意义,可以为苦丁茶冬青及其近缘种的划分提供可靠的依据。根据叶表皮微形态特征的研究结果,不支持霍山冬青与华中枸骨的合并。结合分子生物学证据与叶表皮特征微形态特征的研究结果,形态居间种质材料的冬青属苦丁茶类型应为五棱苦丁茶(Lpentagona)的变种。
光学显微镜和扫描电镜研究结果表明,7种冬青属苦丁茶植物的叶表皮微形态特征具有一定的相似性,如气孔器均位于下表皮,每种植物均有正常气孔器和大气孔器,气孔器类型以环列型为主,其次为双环列型；其差异性体现在叶表皮细胞的垂周壁形态和气孔外缘角质层纹饰等方面。霍山冬青叶表皮微形态与华中枸骨存在着较大区别。
5.观察了不同地域的枸骨和四种性别特征材料的苦丁茶冬青的正常与异常花药与花粉的微形态学特征,同时还研究了大叶冬青、贡山冬青和猫儿刺的花粉特征。研究结果表明,花药纹饰大体相似,但花粉外壁纹饰和萌发沟、花粉体积在种间和种内具有较大差异。不育或败育花粉则在萌发沟深度和长度方面与正常花粉具有较大差异。根据花药与花粉微形态学研究结果,说明花药特征对冬青属系统分类意义不大,但花粉的特征对于冬青属植物的分类具有一定的价值。
扫描电镜研究结果表明,所研究的五类材料其花药纹饰大体相似,都具有不规则脊纹并间有条纹,种间区别不显著；花粉均为三沟型,外壁纹饰均具疣状或钉状颗粒。不同物种间、不同地域的枸骨与不同性别特征的苦丁茶冬青其花粉体积大小指数、纹饰颗粒密度、萌发沟长度和宽度及其比值等特征都具有多样性。此外不育或败育花粉微形态体现在萌发沟深浅和长度不一,具有一定的生态学意义。
6.染色体核型分析结果表明,4种冬青属苦丁茶的核型可分为两类,核型分析的研究结果有助于研究冬青属植物的亲缘关系。
通过酶解离染色后显微镜观察,发现苦丁茶冬青、大叶冬青和形态居间类型种质材料其核型属于2B类型,五棱苦丁茶属于3B类型。就核型分析结果而言,形态居间类型种质材料的核型更接近于苦丁茶冬青。
7.冬青属苦丁茶化学分类学聚类分析研究结果显示,五棱苦丁茶与苦丁茶冬青归为一类,这表明化学分类学与形态数量分类学研究结果基本一致；同时也显示,苦丁茶冬青在化学成分方面具有一定的多样性,这种多样性与其分布地域有关。
根据RP-HPLC数据和NTsys软件进行了化学分类学研究。六种冬青属植物中色谱峰共有峰数目有6个,另外在各个物种色谱图中,作者还发现了一些特殊峰。根据聚类分析的结果,五棱苦丁茶(S12,S13)与苦丁茶冬青(S2)种质材料聚类在一起,其亲缘关系最近,但起源于广西马山石灰岩地区的苦丁茶冬青(S1)种质材料单独聚为1类,其所含有的化学成分种类及含量与其他地域苦丁茶冬青的差异很大。
8.六种冬青属苦丁茶的叶片因种质材料的起源地域,野生来源与栽培条件以及叶片储藏措施的不同,其绿原酸含量存在明显差异。
基于超声提取和RP-HPLC基础上的绿原酸含量研究结果表明,新采集的成熟功能叶经过低温干燥后,其所含绿原酸含量由大到小依次为丁茶冬青>霍山冬青>大叶冬青>五棱苦丁茶>华中枸骨>枸骨；干燥的成熟功能叶放置2月后绿原酸含量显著降低；在苦丁茶冬青野生材料不同发育阶段的叶片中,大部分材料以老叶绿原酸含量最高；一般来说,苦丁茶冬青野生材料叶片中绿原酸含量要高于栽培材料,种间或种内绿原酸含量存在较大的差异,这可能与种质材料的起源地域有关。
9.根据叶总黄酮含量测定结果,并结合绿原酸含量的分析结果,可以认为霍山冬青是一种良好的苦丁茶种质资源；利用幼嫩叶加工成苦丁茶具有其科学性。
在超声提取、络合反应及紫外分光光度法,以芦丁为标准的含量测定结果表明,就5批次栽培材料的功能叶总黄酮的平均值而言,苦丁茶冬青(3.96%)>五棱苦丁茶(3.67%)>霍山冬青(3.63%)>枸骨(3.04%)>大叶冬青(2.64%)；低温干燥(50℃)放置2月后的叶片总黄酮含量明显降低；栽培条件下的苦丁茶冬青,其成熟叶片中的总黄酮含量平均值要高于野生植株成熟功能叶所含量；就所有个体总黄酮含量而言,霍山冬青最高(7.39%)；幼嫩叶的总黄酮含量具有较高的水平。
10.基于红外光谱分析的研究表明,利用红外二阶导数谱图可以将不同物种的冬青属苦丁茶植物和不同来源的苦丁茶冬青种质材料快速和有效地加以区分。
一维傅里叶中红外光谱分析表明,不同物种的冬青属苦丁茶植物和不同来源的苦丁茶冬青种质材料不易区分,有大量的共同的吸收峰,特征峰相对较少。通过对指纹区二阶导数图谱变换,将重叠的光谱分开后,除共同的吸收峰外,不同物种的冬青属苦丁茶植物和不同来源的苦丁茶冬青种质材料叶片红外光谱图吸收峰位置、数目和吸光度显示出了明显差异,利用此差异可以对上述物种或不同来源的种质材料进行鉴别。利用红外光谱聚类分析表明,形态居间类型种质材料与五棱苦丁茶亲缘关系较近,霍山冬青与华中枸骨亲缘关系相对较远。
11.基于SRAP-PCR分子标记的聚类分析结果表明,供试的6种冬青属苦丁茶可分为两大类,与其他分子标记的实验结果基本一致。同时SRAP分子标记实验结果还显示供试的苦丁茶冬青不同的种质材料可分为两大类,这己研究结果为探讨苦丁茶冬青的基因型分类提供了可靠的科学证据。
在本研究中,苦丁茶冬青、五棱苦丁茶和大叶冬青聚为一大类,枸骨、华中枸骨和霍山冬青聚为一大类,这与传统的根据其叶形的分类结果完全一致。也与其他分子标记实验的结果相吻合。因此,SRAP分子标记用于冬青属苦丁茶植物分子系统学研究以及种级水平和种下水平分类鉴定是完全可行的。另一方面,根据聚类分析结果,不同来源的供试苦丁茶冬青种质材料还可分为2个类群,每个类群又可分为若干小类。这说明苦丁茶冬青存在比较丰富的遗传多样性。
本论文在综合分析各种研究手段的实验结果基础上,对冬青属苦丁茶植物的系统亲缘关系及优良种质资源选择进行了探讨。
"Kudingcha" was the general name for a large class of Chinese folk tea beverage plants. There were two types of "Kudingcha" with a large impact at home and abroad among of them. One type of "Kudingcha" was made of species from Aquifoliaceae with the representative species of Ilex kudingcha. The other type of "Kudingcha" was made of species from Oleaceae with the representative species of Ligustrum robustum. This study was limited to the species of Ilex L.(Aquifoliaceae) used as "Kudingcha". The Aquifoliaceae origins of Kudingcha were I. kudingcha and its related species ncluding I.latifolia, I.pentagona, I.huoshanensis,cornuta and I.centrochinensis. Among of six species,the largest of the planting area,yield and economic value was I. kudingcha. There were many factors influencing the industry development and promotion of Kudingcha from Ilex species, and the two key factors were their botanical origin and quality of germplasm resource. But Ilex L.was a groups with more difficult classification, and their had a controversial among different scholars on whether there should merge I. kudingcha and I.kaushue; I.huoshanensis and I.centro-chinensis. In addition, intermediate species with indecisive systematic position (I.sp.) have been revealed by Kudingcha Research Institute of Hainan University, and ts morphological characteristics was intermediated between I. kudingcha and I.pentagona. It have brought a serious influence on sustainable development for Kudingcha. In order to provide scientific basis for solving systematic problems, we have studied the systematic of I. kudingcha and its related species from the perspective of plant morphology, anatomy, micro morphology, numerical taxonomy, chemotaxonomy, infrared spectral analysis, molecular biology. And in order to provide theoretical support for the development and utilization of germplasm resources, Author also have evaluated the germplasm of I. kudingcha and its related species by using methods of RP-HPLC, ultrasonic extraction with standard of chlorogenic acid and rutin. The main results were as follows.
1. Author have observed plant morphology from six Ilex species used as kudingcha. The results showed that stem form of the studied species had five prismatic and circular form. There were abundant diversity for I. Kudingcha in gender and number of fruit, perianth,and locules nuclear of fruit. The above characteristics could have auxiliary meaningful for classification and genetic diversity for Ilex species used as Kudingcha.
Through field observation, author have found that, stem form of I.pentagona was five prismatic and other five species were circular form. The main number were four in number of fruit, perianth and locules nuclear of fruit. In terms of gender, the main gender were unisexual flowers, but there were polygamous phenomenon.
2. Author have observed the anatomy structure of stem and petiole from six Ilex species used as Kudingcha. The results showed that stem structure similar to each other.The differences were the width and density of wood ray, medullary morphology, and the ratio of pith and secondary bundle width.Petiole anatomical structures of the six aboved species are similar to each other too.The difference lies in the form of vascular bundle, and they can be divided into two categories.
Author found that the largest proportion of stem pith width among six species was I.huoshanensis according to the anatomical structure of stem and petiole under the fluorescence microscope. Medullary morphology of stem from I.cornuta, I.centrochinensis was four square, and the other four species were round or nearly round; The species with most wide in wood ray was I.kudingcha, The second was I.latifolia and I.pentagona; The species with regular half moon petiole xylem are I.cornuta, I.centrochinensis I.huoshanensis, and I.latifolia.irregular one are I.pentagona and I. kudingcha.
3.Results of research of stem and leaf morphological quantitative taxonomy of six Ilex species showed that there have similar to the results of petiole anatomy classification, and they could be divided into the same two categories.
The reults of cluster analysis of six Ilex species used as Kudingcha could be divided into two categories based on26morphological variable from stem and leaf.The first class were I.kudingcha and I.pentagona, second for I.latifolia, I.huoshanensis, I.cornuta, I.Centrochinensis.incipal component analysis (PCA) showed that the first principal component mainly reflects the blade symmetry, petiole length, the ratio of distance of base to largest leaf width and leaf length.
4.The results proved that the micro-morphological characteristics of leaf epidermis of Ilex L. had taxonomic significance at the species level, it could be used as reliable evidence in distinguishing I.kudingcha with its related species.There should not be treated I.huoshanensis and I.centrochinensis as the same species according to their micro-morphological characteristics of leaf epdermis.Combined with the evidence of molecular biology and the characteristics of the leaf epidermis, intermediate types should be treated as varieties of I.pentagona.
Micro-morphological characteristics of leaf epdermis for I. kudingcha and its six relative species were examined with an optical microscope and SEM. Micro-morphological structure of leaf epidermis for seven Ilex species as Kudingcha had a certain similarity such as the stomata at lower epidermis.According to the size of stomata, it could be divided into two typies of stomata:large stomata and common stomata.Most of stomatal type were cyclocytic.There also had some differences in leaf epidermis characteristics, such as leaf epidermal cells and stomata anticlinal walls form, the outer edge of the cuticle ornamentation of stomata, etc. And there have a big difference in leaf epidermal morphology between I.huoshanensis and I.centrochinensis.
5. The micro-morphological characteristics of different regions of I.cornuta and four gender characteristics of materials of normal and abnormal anther and pollen of I.kudingcha have been observed. At the same time, the pollen characteristics of I. latifolia,I. hookeri and I. pernyi have also been studied.The results showed that, the anther ornamentation of studied materials were similar to each other. But there had a great difference in the pollen exine sculpture, germinal furrow and pollen size among interspecific and intraspecific level.There had a larger difference between infertility or sterility and normal pollen in germinal furrow depth and length. According to the anther and pollen micro-morphological characteristics, anther characteristics was little significance in classification of Ilex species, but the pollen characteristics had a certain value for the classification of Ilex species.
The results under SEM was showed that, the anther ornamentation was similar to each other among of studied materials. The anther had irregular ridges and striped, cxine sculpture of pollen had verrucous particles and there was no significant differences in anther among Ilex species. Pollen had three germinal furrow. And the exine have ornamentation with verrucous or nail shaped particles. It was diverse in pollen size index, particle density, length and width and its ratio of germinal furrow characteristics among different species, different regions of I.cornuta and different gender characteristics of I. kudingcha. In addition, the characteristic of morphology of infertility or sterility pollen were irregular length and depth of germinal furrow, It could help to clarify some ecological significance.
6.The results of karyotype analysis showed that the four Ilex species could be divided into two classes, and it could help the study of phylogenetic relationships among Ilex species for karyotype analysis.
By using enzymatic dissociation and staining, the results showed that karyotype of I.kudingcha, I.latifolia and intermediate type(I.sp) belongs to2B type, I.pentagona belongs to the type of3B under the microscopy, and it also showed that the form of intermediate type(I.sp)close to that of I.kudingcha according to the feature of karyotype.
7. The results of cluster analysis based on chemotaxonomy showed that I.kudingcha and I.pentagona belonged to the same class, it showed that the chemotaxonomy and morphological quantitative taxonomy have the same results; At the same time it showed that I.kudingcha had a certain degree of diversity in chemical composition, and it may be related with its geographical distribution.
Author have found six common peaks according to chemical taxonomy research based on chromatographic data from RP-HPLC method and Ntsys software. In addition, we also have found some special peak from all chromatograms in six species.According to the results of clustering analysis, Ⅰ.pentagona,(S12, S13)and I.kudingcha(S2) clustered together, the two species had the closest relationship.But I.kudingcha (S1) which come from Guangxi Province, Mashan limestone areas have clustered into one categories alone.There have a big difference both in chemical components and contents combared with the other regions of the I.kudingcha, It may be related to that germplasm materials may have formed a chemical type.
8. Owing to different geographical origin of germplasm, wild and cultivation conditions, storage measures of leaves for six species of Ilex L., there were obvious differences in the content of chlorogenic acid.
The content of chlorogenic acid of fresh mature leaves of six species from high to low is I.kudingcha> I.huoshanesis> I.latifolia> I.centrochinensis> I.pentagona> I.cornuta which have been determined by ultrasonic extraction and RP-HPLC method.; and the content of fresh leaves was much larger than that of the dried up to two months; for content of chlorogenic acid of different developmental stages of wild I.kudingcha samples, old leaves with infected> mature leaves> young leaves.The mean content of chlorogenic acid in the mature leaves of the wild was larger than that of cultivation one for I.kudingcha.There was much difference among six Ilex species and intraspecific of I.kudingcha according to chlorogenic acid content which may be related to the origin of species.
9.The results showed that I.huoshanesis was a better germplasm resources combined with content determination of both the chlorogenic acid and rutin, and it had a scientific significance that people used the tender leave as materials of Kudingcha.
The content of total flavonoids (rutin) from5species of Ilex L.used as kudingcha tea were determined using ultrasonic extracti, complexation reaction and UV spectrophotometry.On the average of value of total flavonoids from5batches of planting materials, I.kudingcha (3.96%)> I.pentagona(3.67%)> I.huoshanesis (3.63%)> I.cornuta (3.04%)> I.latifolia (2.64%); and total flavonoids in the leaves of placed after two months at low temperature dried has decreased significantly; On the average, the content of total flavone content of cultivation of mature leaves of I.kudingcha was significantly higher than that of wild mature leaves.The content of total flavonoids of I.huoshanesis was highest (7.39%) in all individuals from five species; Content of total flavonoids in young leaves maintained a higher level.
10. The results of infrared spectrum analysis of germplasm materials from I.kudingcha and its relatived species based on the second derivative spectra showed that Ilex species used as Kudingcha and different sources of I. kudingcha could be quickly and effectively distinguished with each other.
The one-dimensional fourier infrared spectrum analysis showed that, germplasm materials from Ilex species used as kudingcha and different sources of I. kudingcha were not easy to distinguish and there were a lot of common peaks. The number of characteristic absorption peak was relatively small. The overlapped spectra could be separated by using second derivative spectrum transform of the fingerprint region, In addition to a common peak, it showed obvious differences in IR absorption peak position, number and absorbance among of different species and germplasm materials with different sources of I. kudingcha. Thus it could be identified to those species. The results of clustering analysis by using the infrared spectral have been showed that intermediate species (I.sp) was close to I.pentagona in phylogenetic relationship,and I.centrochinensis had a relatively distantly relationship to Lhuoshanensis.
11. The results of cluster analysis based on the SRAP molecular markers showed that six Ilex species used as Kudingcha could be divided into two categories, which were consistent with experimental results on the basis of leaf morphology and other molecular marker. At the same time, the research results showed that Lkudingcha could be divided into two categories, which provides reliable scientific evidence for the genotypic classification of Lkudingcha.
In this study, I.kudingcha, I.latifolia, I.pentagona had been clustered into one category, I.cornuta, I.centrochinensis, Lhuoshanensis have been clustered into another category, it was consistent with the traditional classification according to its leaf morphology and other molecular marker results. Therefore, it was feasible that SRAP molecular markers could be used as systematics research of identification In the level of species and subspecies.On the other hand, according to the results of clustering analysis, Lkudingcha also could be divided into2categories, and each category can be divided into several small groups.It showed that. I.kudingcha had greater genetic diversity.
Plant systematic and excellent germplasm resource selection among of I.kudingcha and its related species have been discussed on the basis of comprehensive analysis of the experimental results from various research methods.
引文
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