Genotyping Of Leymus Chinensis Based On Microsatellite Markers

Leymus chinensis is a perennial grasses which is widely distributed in the Eurasian steppe.As the king of pasture,Leymus chinensis is widely distributed in the northeastern part of China and the grassland area of eastern Inner Mongolia.It has both ecological value and economic value.It is an important dominant plant species in temperate grassland in China.Leymus chinensis not only has high yield,but also has strong adaptability.It has the characteristics of drought resistance,cold resistance,salt tolerance and so on.Therefore,in the long-term adaptation and evolution process,it has different degrees of differentiation in morphological structure,physiological and biochemical reaction and DNA level.Finally,different types of Leymus chinensis were formed.In this experiment,different genotypes of Leymus chinensis were used as research objects.SSR molecular marker technology was used to classify different genotypes of Leymus chinensis from DNA level,which not only provided a theoretical basis for identification and molecular marker-assisted breeding of Leymus chinensis.It also provides a theoretical basis for the development of molecular markers,molecular evolution and quality breeding for Leymus chinensis.50 pairs of primers suitable for Leymus genus finally obtained 15 pairs of PCR amplification for Leymus chinensis microsatellite.The optimum annealing temperature ranged from 56°C to 69°C,and the allele loci of each pair of primers were CAC,GCC,GA,CAA,TC,AGT,TCT,CAA,CA,TC,CGG,CTG,ACACA,GTC,AG.It was finally determined that the 15 pairs of primers were able to amplify the bands with clear and reproducible bands for the 12 genotypes of Leymus chinensis individuals and conformed to the size range of the expected fragments.After 15 pairs of primers 5’-FAM end fluorescence modification,the optimum annealing temperature range of each pair of fluorescent primers was 60°C~69°C,and the PCR amplification results of 15 pairs of primers were successful,which can be used for capillary electrophoresis detection.By capillary electrophoresis,two pairs of primers,GMW382 and Ltc1570,were too small(about 80 bp),the synthesis was not good,the fluorescence signal was weakor even no signal,and the electropherogram was not detected.The remaining 13 pairs of fluorescent primers could be used for genotyping of Leymus chinensis.According to the electrophoresis pattern obtained by amplification,a total of 45 alleles were amplified,and each primer could detect 1-7 alleles,with an average of 3.46 alleles per pair of primers,and different genes.The molecular weight of the amplified band obtained is between 80 and 200 bp.In this experiment,based on the principle of few hetero peaks,combined with allele and PIC screening criteria,four pairs of core primers were selected,which are also the most effective core primer combinations Ltc0157,Ltc0621,Ltc1100 and GDM068,which are used to distinguish and identify 12 genotypes of Leymus chinensis..The genotypes 2-TM,3-LN,4-CM and 9-CL were distinguished by the primer Ltc0157,and the genotypes 6-GY and 8-LBwere distinguished by the primer Ltc0621,and the primer GDM068 distinguished the genotypes 7-ZSand 10-HM,finally genotypes 1-DQ,5-ZL,11-EE and 12-ZK were distinguished by primer Ltc1100.In addition,according to the data analysis alternatives,six pairs of core primers Ltc1100,Ltc0157,Ltc0621,Ltc1179,Ltc0520 and GDM068 were selected to classify 12 genotypes.