Development Of SSR Markerrs And Genetic Dicersity Analysis In Puccinia Helianthi Schw.

Puccinia helianthi Schw.is a common and damaging disease of sunflower all over the world.This study based on the new generation of Illumina high-throughput sequencing P.helianthi transcriptome data.We searched the P.helianthi SSR loci using the bioinformatics software,analysized composition,distribution and characteristics.SSR primers were designed from transcript sequence contained SSR loci by primer5.0 software.The optimal conditions for SSR-PCR were explored basing on the experiments.Genetic polymorphism analysis and the division of the group were carried out on the 40 P.helianthi varieties.?.SSR in P,helianthi transcriptome were characterized with the MISA software based on Perl platform.A total of 1815 simple SSR were discovered from 1728 sequences.Among all 192 SSR motifs,the most frequent motifs were(A/T)n,(AAT/ATT)n,(TTTG/CAAA)n,(TTTTC)n and(ATCCT)n in each type of perfect SSR,respectively.The SSR ranged from 18 bp to 84 bp,averaging 21.34bp.SSR between 18 and 25bp showed the highest ratio among all of these loci,the rest number was 42.The result of Pearson correlation analysis showed that the length of SSR were positive with the RPKM.The expression level of genes containing microsatellites were lower than those not containing microsatellites.Only 649 of 1728 Unigenes containing SSR were annotated to biological process(264),molecular function(587)and cellular component(423)with GO classification.2.To explore for optimal conditions for SSR—PCR(simple sequence repeat anchored polymerase chain reaction)for P.helianthi,the four factors that would affect PCR(DNA templates,primers,dNTPs and Taq polymerase)were optimized using an orthogonal experimental design based on screening of SSR primers.The optimal PCR reaction(25?L)contained template DNA 2 ng,each primer 0.6pL(10mM),dNTPs 2.5?L(10mM)and Taq polymerase 2.O?L(3U/L).PCR amplifications were performed using a iCycler(Biorad)thermal cycler with 5 minutes of predenaturation at 94?,1 minute of denaturation at 94 ?,1 minute 20 seconds of annealing at Tm(annealing temperature depending on primers),1 minute of extension at 72?,and 10 minutes of extension at 72 ?after 35 cycles of the preceding processes.3.A total of 170 primers were designed from SSR markers,30 primers can effectively amplify in P.helianthi.Screening 10 primers as core primers for the analysis of P.helianthi polymorphism.32 polymorphic loci were obtained in total of 35 loci(91.42%),amplification polymorphism loci range from 2 to 4.Taking genetic similarity(GS)was 0.81456 as threshold,40 P.helianthi were divided into three groups roughly in accordance with the geographical distribution:Group 1:eastern Inner Mongolia,Jilin,Heilongjiang;Group 2:central Inner Mongolia,Shanxi,Hebei;Group 3:western Inner Mongolia,Xinjiang,Ningxia.3 groups of P.helianthi were corresponding to humid and semi-humid continental monsoon climate,arid or semiarid inland climate and semi-moisture semi-aridity inland plateau climate,respectively.