Studies On Genetic Diversity Of Genus Fritillaria From Sichuan Province And Effects Of Temperature And Light On The Growth And Development Of Fritillaria Cirrhosa

Genus Fritillaria L are perennial herb from Liliaceae. It comprises about 130 species of geophytic occurring in south temperate zone of the Northern Hemispher. There are 43 species and 19 varieties of Fritillaria in china. According to Flora Sichuanica, there are 15 species and 4 varieties of Fritillaria in Sichuan Province, and it is one of the distribution centre of Fritillaria resources in China. Herba Fritillaria recorded in Pharmacopoeia of the People's Repulic of China (China Pharmacopoeia Committee, 2005), named "Chuan Beimu" in Chinese, are the dry bulbs of F. cirrhosa, F. unibracteata, F. przewalskii and F. delavayi. They are commonly used as antitussive and expectorant medcine. It is one of the common Chinese medicine in china. And some other species (varieties) in genus Fritillaria were also used as Chinese medicine Bulbus Fritiliariae Cirrhosae in Sichuan Province. The natural environments of Fritillaria area from Sichuan Province in high altitude are unique. Currently, Bulbus Fritiliariae Cirrhosae still mainly depends on wild resources. The medicinal plant Fritillaria resources in Sichuan had been badly destroyed because of that retarded growth, excessive collection and worsening habitat. Strengthening environmental protection and introduction and domestication of Fritillaria in Sichuan is imminent. The genetic diversity and the molecular identification were among genus Fritillaria from Sichuan Province were analyzed by using cytology and molecular markers. In addition, this paper also discussed the effect of growth, physiologic indexes, yielding and the contents of alkaloids of F. cirrhosa D. Don under the different temperatures and light intensity. The main results were described as following:1. Cytological studies on ten poputions in seven species and two varieties of Fritillaria from Sichuan, China. The results showed that the chromosome numbers of Fritillaria were 24. The karyological fomula are as follows: F. cirrhosa D. DON, 2n=24=4m+6st+14t; F. delavayi Franch. 2n=24=2m+2sm+10st+10t;F. unibracteata Hsiao & K. C. Hsia(from Zheduoshan poputions), 2n=24=4m+6st+14t(SAT); F. unibracteata Hsiao & K. C. Hsia (from Maoxian poputions), 2n=24=2m+2sm +10st +10t(SAT); Fritillaria wabunesis S. Y. Tang & S. C. Yueh, 2n=24=2m+2sm+8st+12t(SAT); F. przewalskii Maxim. & Batal. 2n=24=2m+2sm+6st+14t(2SAT); F. hupehesis Hsiao & K. C. Hsia, 2n=24=2m+2sm+12st+ 8t(SAT); F. cirrhosa D. DON var. ecirrhosa Franch., 2n=24=4m+12st+8t(2SAT); F. unibracteata Hsiao & K. C. Hsia var. longinectarea S. Y. Tang & S. C. Yueh var. nov. , 2n=24=4m+8st+12t(SAT); F. Dajinensis S. C. Chen, 2n=24=2m+2sm+10st+10t. The karyotypes of the six (varieties) species are reported for the first time. The karyotype of Fritillaria might evolve towords the weakeness of symmetry. It was deduced that Fritillaria populations geographical distrbuted in Sichuan province were more primitive.2. The ISSR marker technique was used to study relationships and genetic polymorphism of nineteen populations in ten species and one varietal species of genus Fritillaria. The results showed that eleven primers were selected from 35 ISSR primers, and 179 DNA fragments were amplified from 19 populations. Of which, 179 fragments were polymorphic (percentage of polymorphic bands was 86.8 %). The genetic similarity among all accessions ranged from 0.569 to 0.855. Clustering analysis showed that the 19 populations of Fritillaria could be distinctively classified into 4 groups. ISSR marker technique is suitable for the genetic diversity of Fritillaria from Sichuan Province. In addition, the cluster result of genus Fritillaria had some relationships with the geographical distribution. Interspecific identifications among the four original species of Bulbus Fritillariae Cirrhosae recorded by pharmacopoeia of China, and between them and the other species of genus Fritillaria from Sichuan Province could not be gained by using ISSR markers technique.3. The internal transcribed spacer (ITS) regions of the rDNA were determined in 19 taxon of Fritillaria were cloned and sequenced. Molecular phylogenetic relationships of the related taxon of Fritillaria were studied. The result showed that the sequence of ITS ranged from 658 to 661 bp in length. The 5.8S subunit of Fritillaria was found to consist of 163 or 164 bp nucleotides, while the length of ITS1 and ITS2 varied from 214 to 216 bp and from 283 to 284 bp. The sequences of ITS (ITS1 and ITS2) were about 1.2～1.7 times more variable than those of the 5.8S subunit. The genetic distances varied from 0 .0020 to 0.0730. Based on ITS spacer analysis showed that F. wabunesis had closest relationships with F. unibracteata var. longinectarea, and the relationships between F. sulcisquamosa and F. hupehensis were farther, relatively. F. sulcisquamosa and F. cirrhosa were farthest relatively. Clustering analysis showed that the 19 populations of Fritillaria could be distinctively classified into 5 groups. The cluster result of genus Fritillaria and the morphological classification have consistency. In addition, the cluster result of genus Fritillaria had little relationships with the geographical distribution. Interspecific identifications among the four original species of Bulbus Fritillariae Cirrhosae recorded by pharmacopoeia of China, and between them and the other species of genus Fritillaria from Sichuan Province could not be gained by using ITS markers technique.4. The segments of chloroplast DNA (cpDNA), namely matK were determined in 19 taxon of Fritillaria were cloned and sequenced. Molecular phylogenetic relationships of the related taxon of Fritillaria were studied. The result showed that the matK ranged from 1743 to 1751 bp in length. The genetic distances varied from 0.000 0 to 0.025 0, with an average of 0.011 0. Based on matK analysis showed that F. przewalskii had closest relationships with F. delavayi; and the relationships within F. thunbergii and F. sulcisquamosa and F. wabunesis were farther relatively. Clustering analysis showed that the 19 populations of Fritillaria could be distinctively classified into 5 groups. The cluster result of genus Fritillaria and the morphological classification have consistency. In addition, the cluster result of genus Fritillaria had little relationships with the geographical distribution. Interspecific identifications among the four original species of Bulbus Fritillariae Cirrhosae recorded by pharmacopoeia of China, and between them and the other species of genus Fritillaria from Sichuan Province could not be gained by using ITS markers technique.5. Using artificial climate controls the growth temperatures(15℃, 20℃, 25℃and 30℃) of F. cirrhosa, the difference of their growth development were observed, the contents of chlorophyll a and b, soluble sugar, MAD, proline of the leavses of F. cirrhosa were tested, and the yielding and the alkaloids contents of the bulbs were analyzed. The growth period of F. cirrhosa under 15℃and 20℃were appropriately extended. The contents of chlorophyll b of F. cirrhosa leaves under four different temperatures are insignificant. The contents of total chlorophyll and chlorophyll a were also insignificantly different under15℃, 20℃and 30℃. The contents of soluble sugar, MAD and proline of F. cirrhosa leaves and the growth ratio, dry weight and content of alkaloids of F. cirrhosa bulb increased with the temperature decreased. Higher temperature is not suitable for the growth of F. cirrhosa. Under the relatively lower temperature, the growth period of F. cirrhosa extended, the bulb can grow properly, and the content of alkaloid increased. F. cirrhosa can improve its cold tolerance by increasing the content of proline and soluble sugar, and it also can maintain the normal content of chlorophyll under the lower temperature.6. The experiment was conducted to study the growth, physiologic indexes, yielding and the contents of alkaloids of F. cirrhosa D. Don under the different light intensity. Using artificial climate controls the growth light intensity (3000 Lx, 6000 Lx, 9000 Lx and 12000 Lx) of F. cirrhosa with temperature of 15℃. The difference of their growth development were observed, the contents of soluble sugar, MAD, proline of the leavses of F. cirrhosa were tested, and the yielding and the alkaloids contents of the bulbs were analyzed. Under the light intensity of 6000 Lx, the growth and development of F. cirrhosa was better, and the dry weight and the alkaloids contents of the bulbs of F. cirrhosa accumulated more. But the dry weight of F. cirrhosa bulb reduced under the lower or higher light intensity. The contents of soluble sugar, MAD, proline of the leavses of F. cirrhosa under four different irradiances are insignificant. The light intensity (6000 Lx) is suitable for the growth of F. cirrhosa.