Characterization Of Microsatellites In Poplar And Willow And Development Of Their Universal Primers

Microsatellite(SSR)has the advantages of co-dominant markers,high polymorphism and easy detection.It is widely used as the molecular markers technology.In this study,we took the genome of willow and poplar as our research target.Whole genome SSRs of Salix were developed,which will provid abundant resources for the study of classification,genetic diversity and evolution in willow.Combining the whole genome SSR markers of populus,we analyzed the sequences of SSR in paralogous and orthologous genes of poplar and willow,respectively.It revealed the trend of poplar and willow genome after their differentiation.In addition,based on the published poplar SSR primer database,we searched the universal primers of poplar and willow,which will lay the foundation for establishing their ge netic system.1.A total of 109972 perfect microsatellites were obtained by analyzing the whole genome sequence.The frequency these SSRs in genome,intergenic regions,introns,exons and UTRs were 1/4799 bp,72.8%,19.2%,4.2% and 3.8%,respectively.According to the analysis of the frequency in different types of SSRs,we found that the frequency of SSRs decreased with the repeated unit lengths,and the shorter repeated unit gains repeats at a higher rate.It revealed that the frequency of different types of SSRs in the willow genome is not random,and the GC content in the repeated sequences is significantly correlated with the gene function.2.We divided the whole genome of willow into 174 windows.The distribution of exonic_SSR was tested by the Poisso n distribution.C lustering and dispersion of SSRs within each chromosome was evaluated by opening sliding-windows along each chromosome.The SSRs were overabundant in 67 windows,whereas SSRs were sparse in 27 windows.The most abundant and dispersed distribution of SSRs occurred on chromosome 16 and chromosome 11,respectively.The total base number of abundant windows accounted for only 11% of the whole genome,and the total number of dispersed windows reached 55% of the genome.It revealed that the distribution of exonic_SSR on the genome was uneven,and a mass of SSRs variation existed in the high repetitive sequence region.Finally,SSR primers in gene regions were designed by the primer3 software,which will provide abundant resources for the study of classification,genetic diversity and evolution in wil ow.3.We analyzed the characterization of SSRs in the paralogous genes of poplar and willow,respectively.It was found that the majority of the paralogous genes in poplar and willow genome originated from the “salicoid” duplication event.During the process of evolution,willow lost more gene copies than poplar,and larger variation of SSRs occurred in willow paralogous genes.It can be deduced that the poplar genome is more conservative than willow after genome divergence.4.Comparing the characterization of SSRs in poplar and willow orthologous genes,it was found that more than 50% of SSRs only exited in one species.The repeat units of these SSRs were analyzed;we found that the most frequent motifs were A/T,which was consistent with the characteristics of the SSRs in coding regions of poplar and willow.The average GC contents of SSRs were 0.22.It was lower than those in exonic regions.Therefore,the SSRs in the orthologous genes were unstable,indicating that the homologous gene will continue to differentiate.5.Totally,8462 universal pairs of primer were obtained by using blastN to align the populus SSR primer database to S.suchowensis genome sequence.The universal primers were divided into three categories:(1)SSRs were not detected in the willow sequences(2)SSRs were detected in the willow and they were same with poplar sequence(3)SSRs were detected in the willow and they were different from poplar sequence.Forty pairs of primers were randomly selected to assess the amplification effciency in 24 willow natural populations,and thirty-six pairs primers were amplified successfully.The results indicated that SSRs had a high transferability in poplar and willow.The PIC polymorphism of 29 univers al primers containing SSRs was tested,and twenty-two pairs had polymorphism.For the second and third type of primers,the PIC value were 30% and 39.8%.3.42 and 3 alleles per pair of SSR primers were detected,and the number of alleles per pair of SSR pr imers could be 2 to 7 and 1 to 4.Totally,71 alleles were detected.