| Red clover(Trifolium pratense L.)is a perennial herbaceous plant of Leguminosae grown in Yunnan, Guizhou, Gansu, Jiangxi, Sichuan and Xinjiang province. However, red clover germplasm resources of China are quite less, a few wild species distributed in Xinjiang, hubei, Sichuan and other provinces. The research revealed phenotypic diversity and genetic diversity( ISSR) of 31 red clover germplasms from Russia and North America, at Yuzhong County, in the middle of Gansu province, to provide a theoretical basis for development and utilization of red clover germplasm resources and new variety breeding. The main results are as follows:(1) 31 red clover germplasm resources were used and 14 traits were measured in terms of morphological characteristics, agronomy traits and productivity. Significant differences were found in growth period, plant height, branch number, leaf area, stem-leaf ratio, regrowth speed and hay yield. The source of the materials also played a role on morphological characteristics and productivity. The materials from America had big leaf, long internode, high hay yield, quick regrowth speed and higher thousand seed weight; Russian materials had more branch number, small leaf, short internode, lower hay yield and small seed. A few materials had super performance on single trait and could be used in production directly.(2)According to principal component analysis, five principal elements with 78.94% cumulative contribution rate were used for comprehensive evaluation of red clover germplasm. The result showed that ZXY2008P-4996 from Russia was the best, followed by Scarlett and Star fireⅡ from the U.S. ZXY2008P-5419 had the lowest score and the poorest adaptability. The 31 red clover germplasm were grouped into 4 clusters by means of principal component index.(3)An orthogonal design was applied to optimize an ISSR-PCR amplification system for red clover, involving four levels of four main factors(Taq DNA polymerase, Mg2+,premier and d NTP). An optimal reaction system was established. The reaction was performed in a 25μL solution containing 40 ng template DNA, 10×PCR-buffer 2.5 μL, 2.5 mmol/L Mg2+, 2.5 U Taq DNA polymerase, 1 μmol/L premier and 0.2 mmol/L d NTP. The optimized reaction program was: 94℃ for 5 min, followed by 35 cycles of 94℃ for 30 s, annealing 30 s, 1 min at 72℃and a final extension at 72℃ for 7 min, then preservation at 4℃. The establishment of the optimized amplification system provides a solid foundation for the analysis of genetic diversity in red clover with ISSR markers.(4)Genetic diversity of 31 red clover germplasm resource was studied using ISSR-PCR molecular markers. 5 primers with strong polymorphism and good repeatability were selected from 20 ISSR primers. Five primers detected 61 bands including 58 polymorphic bands, and the proportion of polymorphic bands(PPB) was 95.08%. The results from POPGENE analysis indicated that the genetic similarity coefficient of red clover germplasm ranged from 0.5902 to 0.9344, the average information index(I) of Shannon was 0.3427, genetic diversity index(H) of Nei was 0.2092 and Effective number of alleles(Ne) was 1.3162, indicating a comparatively rich genetic diversity among the tested materials.(5)UPGMA cluster analysis was used and 31 materials were divided into five types, Kenway from Canada had the highest genetic differentiation and formed an unique group. The majority of germplasm from same origin were classified into the same group, indicating the genetic distance of the tested materials were closely related to their geographical distribution.(6)There were both overlapping and different regarding the results of morphological markers and ISSR molecular markers. Morphological markers mainly revealed the differences among different morphological characters, and most of the quantitative traits were easily affected by the environment; ISSR molecular marker indicated the differences among different genotypes, directly revealed the genetic variation in DNA molecular level and revealed more genetic information. |
