Research On Transcriptome Sequencing And SSR Markers Development Of Salix

In this research, RNA-seq was carried out to produce substantial expressed sequence tags(ESTs) by use of leaves of S. babylonica and S. suchowensis through454GS FLX platform. Onthe basis of database, a set of SSR markers were developed; and the transferability of SSRmarkers of salix was analyzed between different species and hybrids. The main results are asfollowed；1.454GS FLX platform and Titanium regent were used to produce EST from the leaves ofS. babylonica and S. suchowensis. A total of547104ESTs(280074in S. babylonica,267030in S.suchowensis) with an average read length of415bp were generated. The ESTs of S. babylonicawere assembled into40271Unigene(4701Contig and35570Singlet); the ESTs of S. suchowensiswere assembled into55083Unigene(7793Contig and47290Singlet). All Unigenes werefunctionly categorazied into GO(Gene Ontology) categories and anaysised metabolic pathwaysinto KEGG respectively.550different expressed genes were found between S. babylonica and S.suchowensis, these genes were functionly categorazied into GO(Gene Ontology) categories.2. The analysis found that MISA was the most fast and effective software which find SSRfrom Unigene of S. babylonica and S. suchowensis. Based on transcriptome sequencing EST dataresource,4521SSR were found in all Unigenes of salix using MISA software (1752SSR in S.babylonica and2949SSR in S. suchowensis). Then, use the Primer5software to design primers,a total of1288pairs of primer were designed (459in S. babylonica and729in S. suchowensis).Finally, there were1082pairs of SSR primers successful amplification in their parents by PCRand agarose gel electrophoresis,424pairs in S. babylonica and658pairs in S. suchowensis).3. In order to explore the variation trends of genic SSRs in different tree species, fiftythousand Unigene were analyzed of salix、Populus and Eucalyptus respectively in this study. Theresults showed that the percentage of Unigene containing SSRs was similar in poplars andeucalyptus, accounting for19.3%and19.9%respectively. This ratio was significantly lower insalix, only accounting for11.4%. A common phenomenon observed in the three tree species wasthat the triplet repeats were the dominant microsatellites in the investigated Unigene sequences;the frequency of microsatellite gaining or losing repeat unit/units decreased with increment in therepeat motif lengths of different types of microsatellites.4. The transferability of salix SSR primer was analyzed based on100pairs of S. babylonicaSSR primer and100pairs of S. suchowensis SSR primer, which amplified in16varieties of salix.Results showed that transferability of SSR primer of salix to the other16varieties of salix isbetween70%and99%. Among them, S. matsudana And S. hypoleuca have the besttransferability in100pairs of S. babylonica SSR primer, as high as93%, but was only74%in S.suchowensis. Transferability of S. suchowensis100pairs primer in S. suchowensis×S.leucopithecia was99%，and the transferability of S. babylonica was only63%. We found thattransferability of SSR primer of S. babylonica in trees willow was better than in Shrub willow and the percentage of effect S. suchowensis SSR primer in Shrub willow was the better than intrees willow through Comparative and analysis.