| White clover(Trifolium repens L.)as legume(Leguminosae)clover(Trifolium)perennial herb with significant agronomic characters,is one of the most widely used leguminous forage cultivation in the world,and which distributed in the most of the provinces and autonomous regions of China,especially cultivated in the large area of the south of Yangtze river,white clover is also recommended as the main leguminous forage for the vast southern regions of our country.Moreover,because of its graceful beauty,good appreciation and well adaptability,white clover is extensively applicated in the field of environmental afforestation,water and soil conservation and ecological construction.In this study,genetic diversity and relationship of 71 accessions of rhizobia isolated from wild white clover obtained from six sampling sites including Ya’an,Kangding,Luding,Xichang,Chengdu and Leshan in Sichuan province of China were analyzed by SRAP markers.Based on the result of 12 effective rhizobia strains preliminary obtained from hydroponics and sand-cultures,with commercial clover rhizobia ACCC18016 for reference strains,white clover(L.cv.’Ladino’)as the host plants.Further screening by soil culture methods and artificial simulation of the drought stress for the advantage of drought resistance strains induced by polyethylene glycol 6000(PEG-6000).The major results were listed as follows.1.A total of 292 bands were detected by using 23 primer pairs,of which 279(95.55%)were polymorphic loci.The average percentage of polymorphism information content(PIC),band informativeness(BI),resolving power(RP),marker index(MI)for each primer combination were 0.341,0.429,6.226 and 4.507,respectively.Genetic similarity(GS)among all accessions ranged from 0.493 to 0.962 with an average of 0.673.Based on the UPGMA cluster analysis,71 accessions were divided into three groups at GS=0.59,and the accessions from the same or similar geographical origin showed the relative centralization results.The significant difference(p<0.001)among genetic variation were observed according to the molecular variance analysis(AMOVA)85.05%between geographical groups and 14.95%within geographical groups.2.Soil potting experiment inoculated with rhizobia showed that plants had the better status with higher plant height,luxuriant growth,large and deep-color leaves,which were just the opposite of the controls without inoculation.The best symbiotic matching existed within YA21 and XC09 rhizobium strains.When compared with controls,the physiological index such as plant height,fresh weight,dry weight and chlorophyll content of vaccination strain YA21 plants were enhanced by 62.89,34.65,86.05 and 33.27%,respectively,which were close to the commercial strains ACCC18016 and exhibited the obvious advantage than the rest of strains.After inoculation with XC09 strains,whiter clover plants maintained rich crude protein and high nutritive value,and the content of crude protein was significantly raised by 32.28 and 4.74%relative to controls and strains ACCC18016 separately.While inoculation with rhizobia had no significant effect on the whiter clover root length and the content of crude fiber.3.The growth and reproduction ability of white clover rhizobium was reduced rapidly with the enhancement of drought stress intensity,and all tested rhizobium was very sensitive to water defict with a diverse ability of drought tolerance,srtains YA03,YA05,KD04 and LD02 exhibited a greater tolerance to drought stress,of which YA03 and YA05 maintained the significant advantage of tolerance under severe drought stress,while KD04 and LD02 strains were more suitable for growing in the mild drought stress environment.These results suggest that SRAP marker with a good ability to identify the rhizobia of white clover,and which to be an effective means of genetic diversity analysis,it also revaled abundant genetic diversity among all selected white clover rhizobium strains.Meanwhile,the highly effective stains of YA21 and XC09 as well as YA03,YA05,KD04 and LD02 for drought tolerance strains had been obtained form the preliminary screening. |
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