| Development and screening of a set of comprehensive features well and have gene-r al purpose core SSR primers get significant for maize germplasm identification,heterosis group division,analysis of genetic diversity,variety purity and authenticity of detection,DNA fingerprint database building and other research.However,existing maize SSR primer s are insufficient to meet the needs of scientific research and test,In the standard for corn 40 primer existing problems:a part of primers poor stability,missing data more;series peak and other abnormal peak go against automatic classification for software,need artificial correct-ion,heavy workload;2bp repeat primers is more,is not easy to realize data sharing between different electrophoresis platform;single PCR waste time and energy,the advantage is smaller compared with multiplex PCR.For the reasons come up with perfect SSR,select markers such as :basic groupā„3,primers flanking sequence conservatism powerful,can effectively remove abnormal peak contain series peak,at least can compose5 multiplex PCR or 10 multiplex electrophoresis,improve experiment efficiency,be beneficial to realize data sharing between different electrophoresis platform,for the following research:Firstly,the study obtain a scheme about suitable maize cultivar identification,and get perfect SSR primers by a lot of primers screening and consist multiplex PCR,The primer screening major could be divided into two stages,the first stage,in order to determine a set of suitable solution f-or SSR primers evaluation,In this study,111 pairs of new developed maize SSR primers as the research object,Using the 95 inbred lines and 27 sets of moth-er-father-child trios,separately using two kinds of DNA analyzer to screening primers about Qiagen and ABI3730,screening 9 pairs of primers with high polymorphism,good stability and suitable for automatic classification;determine suit-able scheme about SSR primers screening,provide high efficiency screening scheme about primers design;evaluate and choose in future,and lay a foundation f-or improving excellent SSR primers selected ratio.Based on the study of the screening process,established a set of suitable for SSR primers screening evaluation scheme.The second stage of screening work,according to the previous experiment summarize and reset the primers development parameters,both of the primers quality and selected ratio get larger improvement.Primers selected ratio is higher,sample amount is larger,in directly use ABI3730 fluorescence capillary electrophoresis screening primers,can obtain primers expression on large samples rap-idly,effect is intuitive.First using 22 materials of first screening,selecting expression excellent primers enter into second screening,22 materials include three sets of mother-father-child trios,and other selfing lines genetic relationship is fart-her,representativeness is stronger.Using 93 selfing lines proceed secondary screening,choose 90 primers about high polymorphism,good stability and suitable automatic genotyping.Analysis the selected primers by means of bioinformatics,acquire primers interaction coefficient,according the principle about chromosome distribution on average and fragment differ about 20 bp,consist of 5 multiplex PCR,achieve 10 multiplex electrophoresis,at present have compose 5 multiplex PCR amount to 8,10 multiplex electrophoresis amount to 4.In the process of primer screening,select performance imperfect SSR primers,such as N+1 peak,nonspecific PCR,double peak,do not conform Mendeian genetic law and other phenomenon,describe and analysis the forming reason;Choose part of primers and analyze the cause by means of sequencing,find 2 primers have InDel locus before simple sequence repeat,redesign primer about SR26829 and achieve satisfactory effect. |
