| Kentucky bluegrass(Poa pratensis L.)is a commonly used grass that possesses excellent quality,as well as a complex genetic background and reproductive patterns.Along with the global warming in recent years,high temperature stress is one of the most important reasons that prevent the further promotion of Kentucky bluegrass.In this study,a total of 89 germplasms were collected,of which 50 were imported varieties,5 were Chinese breeding varieties,and 34 were wild materials.At first,73 materials were investigated phenotypes and analyzed the genetic diversity by random amplified polymorphic DNA(RAPD),simple sequence repeat(SSR)and sequence-related amplification polymorphism(SRAP)marks respectively.Then,85 of total materials were submitted to heat stress(40 ℃ /28 ℃,day/night,16h/8h)in an artificial climate chamber.Before and after heat stress treatment,plant height and state score were measured respectively.According to the tolerance days and status,nearly 1000 plants were ranked by their heat resistance.Finally,15 heat tolerance materials and 15 heat sensitive materials were selected from the population.One plant per materials was selected to construct the heat-tolerance or heat-sensitive DNA bulk.SRAP and SSR markers related to heat tolerance were screened by the bulked segregant analysis(BSA)method.According to the sequences of the heat-related fragments,sequence characterized amplified regions(SCAR)primers were then designed.Furthermore,the SCAR markers were tested in 30 clones and verified in heat testing populations.The main findings were as follows:1)High levels of polymorphisms were observed in RAPDs(91.3%),SRAPs(91.8%)and SSRs(94.5%),respectively.The dendrograms that were generated from three kinds of molecular markers revealed a general similarity for the positioning of the majority of materials.Most of the imported varieties were gathered together,and four domestic varieties(‘KBG04’,‘KBG03’,‘Huhe 2’and ‘Zahe’)were also gathered together.However,the distributions of domestic and foreign wild materials in the four cluster maps were not same exactly.Further analysis demonstrated no significant association between geographical origin and molecularmarker clusters in the wild materials.Combined with the seedling phenotype identification,it seemed as though there was no significant relationship between agronomic characterization and marker-based clustering in these materials,except for in the case of leaf color.2)The growth rates and state scores of all the materials showed an obvious downward trend under heat stress.And the decreasing rate was accelerated gradually along with the increase of stress time.According to the ranking of heat resistance of85 Kentucky bluegrass materials,the most heat tolerance materials were almost imported varieties,while the most heat sensitive materials were wild materials.The domestic varieties ranked in the middle.3)88 SSR and 304 SRAP primer pairs were used to screen the different bands between the two DNA bulks and 30 individual plants.According to the cluster analysis of polymorphic bands and heat tolerance trait,the most related bands were selected and converted to SCARs.A total of 24 pairs of SCAR primers were designed and verified in the 30 individuals of the two pools.The polymorphic bands amplified by 19 SCAR markers were approximately identical to those of its corresponding SRAP/SSR markers.To a certain degree,11 SCAR markers were related to the heat tolerance in the heat testing populations.However,with the decrease of heat tolerance,the bands did not decreased gradually.The ratio of band appearance was obviously decreased only in the most heat sensitive materials. |
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