Comprehensive Evaluation On The Agronomic Traits And Construction Of The DNA Fingerprinting Using SSR Markers For Annual Ryegrass(Lolium Multiflorum L.)Varieties(Strains)

The variety test was carried out among13Lolium multiflorum varieties or new lines in phenological period, regrowth speed, resistance, herbage yield, the ratio of stem to leaf and nutrition facts using Changjiang No.2, Tetragold, Aubade and Splendor as the check varieties. Through comprehensive analysis and evaluation of the agronomic traits from2010to2012in Ya’an and Hongya, we selected some new Lolium multiflorum varieties with good quality and high yield which was suitable for cultivated in southwest China.By use of two national authorized varieties, we established the scientific sampling strategy which was very important to the authenticity and the efficiency of molecular genetic diversity analysis of Lolium multiflorum varieties using SSR markers.Based on the scientific sampling strategy, DNA fingerprinting of the21Lolium multiflorum varieties or new lines was established successfully.The work laid the foundation for the large DNA fingerprinting datebase building as well as declaration of the new variety and varieties protection of intellectual property rights. The main research results were as follows:(1) The new lines Florida4N, Z3and Z6were the most excellent and stable on the yield,while Z3had obvious yield advantage in spring and winter observed in2011and2012. If only analysis of the first three bifurcation grass,the hay yield of Z3achieved12994kg/hm2during the two years on an average, increased with the range from11.50%to15.66%comparing with the check. The fresh yield of Z3achieved98973kg/hm2during the two years on an average increased with the range from9.47%to12.82%comparing with the check. So Z3was Suitable for rice straw, corn-annual ryegrass and some other hay crop rotation.In addition, Z3was very rich in leaves,well-adapted,grew faster in winter achieving at0.4cm/d. During the reproductive period, the height of Z3was significantly higher than the check,especially from late December to the middle of February.(2) Two Annual ryegrass varieties, ChangjiangNo.2and Tetragold, were selected and their bulked DNA samples obtained from5,10,15,20,25and30individual plants, respectively. The result of genetic diversity analysis based on simple sequence repeat (SSR) markers showed that there were no differences in the PIC index among seven sampling methods, whereas PIC reached the highest when bulked plant sample were20. The number of average allele and the alleles’ types of per SSR loc increased along with the plant number in the bulked plant samples increasing, which kept relatively steady when bulked plant samples surpassed20(included20) plants, meanwhile the SSR banding patterns were relatively uniform. The bulked samples20were able to detect the most high frequency (>10%)alleles with excellent reproducibility, if disregard the undetection of rare alleles(<10%). According to the results above,20individual plants was the suitable sampling size in genetic diversity analysis of annual ryegrass based on SSR markers.(3) On the basis of others’and our previous research experience, we took the bulked DNA samples of20individual plants for each group in the experiment. Twelve evenly distributed SSR primer pairs with high polymorphisms and good repeatability were successfully screened out from100candidates to construct the fingerprinting database. Among the21varieties,12primer pairs produced108polymorphic genotypes, the ratio of polymorphism was as high as94.15%and9genotypes were detected by each SSR primer pair on an average with the range from5to22; the polymorphic information content values (PIC) ranged from0.744to0.934with the mean of0.843. The twelve SSR primer pairs with high polymorphism, good repeatability and specification could be used as the core primer to construct the DNA fingerprinting of more annual ryegrass varieties. The genetic similarity coefficient of21accessions varied from0.4956to0.8571. UPGMA cluster analysis of genetic similarity showed that all the materials were clustered into three groups at the genetic similarity of0.68. For distinguishing germplasms more simply, through selection of the primer pairs according to its PIC step by step, we found that the primer15-08C could distinguish all the germplasms in the experiment separately. At last, we constructed the standard mode image of DNA fingerprinting of21annual ryegrass varieties by the primer15-08C.Now each germplasms in the experiment had its own fingerprinting (bandtype).