Research On Genetic Variation Of Diploid And Tetraploid In Siraitia Grosvenorii

Siraitia grosvenorii (Swingle) C. Jeffrey, belonging to the genus Siraitia Merr under the family Cucurbitaceae, is a precious and economically important species endemic to southern China and has been cultivated for several centuries. The fruits of S. grosvenorii, called Luohanguo, are used for food, beverages and traditional Chinese medicine. The fruit has evident effects on dry cough, sore throat, extreme thirst and constipation. The major components of Luohanguo are mogrosides, a group of terpene glycosides, estimated to be about 300 times as sweet as sucrose. Moreover, Luohanguo extracts have chemopreventive and antioxidant properties. Its non-caloric properties and effects on the cancer chemopreventive and antioxidant, are being used as sweetening agents for the patients with diabetes mellitus, adiposis, hypertension, heart disease and so on. Mogrosides only exist in flesh, occupying 15% of the dry weigh, whereas seeds constitute 70% of the weight but contain no mogrosides. Thus, seedless Luohanguo will no doubt increase the utilization and extraction rate of mogrosides.Our group isolated a novel male mutant plant from hybrids of'Bolin' and 'ND'. The mutant exhibited growth vigor, larger body and increased organ size compared to wild type. We then used pollen to pollinate 'Nongyuan', and the following year fruits of female hybrids were seedless. In this study, we carried out preliminary studies on karyotype analysis and the molecular mechanism of mutant. The results will provide reference information and resources for molecular breeding of seedless Luohanguo.1. The chromosome numbers of wild-type, mutant and hybrid were 2n=2x=28,2n =4x=56 and 2n=3x=42, respectively. The results showed that wild-type was a diploid strain. Mutant was a tetraploid, and hybrid was a triploid. To our knowledge, this is the first report on karyotype and molecular studied of polyploids in S. grosvenorii.2. A total of 196 SRAP primer combinations were used for DNA amplifications,9 combinations of which failed to amplify banding patterns.189 SRAP primer combinations generated 4573 bands,577 (12.6%) of which were polymorphic while 1998 pairs (87.4%) were identical. Fragment bands obtained were in the size range of 100-800 bp, with a mean of 12.1. There were no apparent differences in the bands of M036 and M038 amplified by most primer pairs. The results generally indicated that the genetic diversity would be quite low between the diploid and tetraploid strains of S. grosvenorii. 3. A total of 196 SRAP primer combinations were used for cDNA amplifications,63 of which failed to amplify any banding patterns. A total of 133 SRAP primer combinations generated 2917 bands,289 (9.9%) of which were polymorphic while 1313 pairs (90.1%) were identical. Fragment bands obtained were in the size range of 100-800 bp. Stable and clearly differentiated fragments were sorted out, cloned and sequenced. Ninety-two differentially expressed fragments were successfully sequenced,77.2% of them were highly homologous to known genes,9.8% were hypothetical genes and 13.0% were possible new genes as they had no significant similarity to known genes. Sequence analysis revealed that most fragments had significant homologous nucleotide sequence to photosynthesis, respiration and stress response genes. These genes included ribulose-1,5-bisphosphate carboxylase/oxygenase, phosphoenolpyruvate carboxykinase, pyruvate kinase, peroxisomal membrane transporter, NBS-LRR type resistance protein, protein phosphatase and others. Functional analysis showed that the gene-encoded proteins were involved in a broad range of biological pathways, including transporters, signal transduction, metabolism, transcription, protein synthesis, development, energy, etc. There proteins play important roles in regulating palnt growth and development and include zinc finger protein, molecular chaperone, mitogen-activated kinase, transcription factor IWS1, transducin, endomembrane protein, porin, cellulose synthase, cytochrome P450, glycosyltransferase, oxidoreductase and other. The results, to some extent, indicated that the tetraploid of S. grosvenorii has more resistance to stress and photosynthesis ability than its diploid relatives. It provided molecular evidences for the phenomenon of polyploids exceeding their diploid relatives based on the phenotypic and biological features.4. With Solexa high-throughput sequencing technology for transcriptome assembly of seeds from F049 and F050,43,891 Unigenes are obtained. There are 17593 Unigenes involving in metabolic pathway. In the DGE analysis of seeds from F049 and F050, 87350 reference tags and 41678 genes are obtained. The analysis shows that mRNA, which is less than 5.5%-5.8%of the types of mRNA, accounts for nearly 76%-77%of the total ammount, but all which accounts for 58%-62% of types, is less than 4.2%-4.5% of total amount mRNA. Statistics on the differentially expressed genes show that, in the F050/F049 genes, there are 1748 genes up-regulated, while 2037 genes are down-regulated.In summary, in this study we carried out preliminary studies on karyotype analysis and the molecular mechanism of mutant strain. The interesting tetraploid mutant contains changes in genome structure and gene expression compared to the diploid strain. As this is the first study on tetraploid of S. grosvenorii, which will provide solid basis for future studies on tetraploid and hybrid triploid seedless Luohanguo. The results will provide reference information and resources for molecular breeding of seedless Luohanguo.