Study On Systematics For Ilex Kudingcha And Its Related Species And The Evaluation For Their Germplasm Resources

"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.