Construction Of High-capacity Transcriptome Library Of Rehmannia Glutinosa Libosch And Exploitation And Identification Of The EST-SSR Markers

Rehmannia glutinosa Libosch., a scrophulariaceous perennial herbaceous plant, is a very important Chinese herbal medicine staple crop in China, which has a very long story and positive clinical effects. However the production of Rehmannia glutinosa faces problems like existing varieties aging, single germplasm resource and poor technology of new varieties breeding, which severely restricted the sustainable development of Rehmannia glutinosa yields. In order to select new varieties which have higher heterosis and variation stability and solve the problem like Rehmannia glutinosa parent resource scarcity, the genetic background of Rehmannia glutinosa variety is obscure and narrow, breeding work blindly repeats, and pharmaceutical personnel are disconnected with medicinal herbs planters and breeders, we need to divide the interspecific genetic distance of different breeding resources of Rehmannia glutinosa drastically on the genetic side. Because Rehmannia glutinosa genome resources are desperately short now, so it’s hard to exploit the genonme SSR marker. We has done the research on the exploitation, identification and polymorphism evaluation of Rehmannia glutinosa EST-SSR marker on the basis of constructing the high-capacity transcriptome library sequences of Rehmannia glutinosa, the main research contents are as follows.Our works was the first time to construct the high-capacity transcriptome library of Rehmannia:Obtained99708transcriptome sequences in the roots,94544transcriptome sequences in the leaves and94479transcriptome sequences when the roots and leaves combined together respectively using Solexa sequencing technology. On this basis, transcriptome library of Rehmannia root tuber, all Rehmannia EST sequences by traditional Sanger sequencing technology and transcriptome library constructed in this study by Solexa sequencing technology were assembled together by a cross-platform joint optimization, a total of87665transcripts were obtained after a series of redundancy reduction processing, low-quality reads and short sequence fragments removal processing; the longest fragment was8009bp. the shortest fragment was204bp and N50was635bp. In case of Rehmannia genomes being vague and unknown, firstly in this study, we acquired the greatest quality, the minimum redundancy and the largest flux transcriptome library in present research on Rehmannia, and provided an important information platform for the medicinal components synthetization, growth mechanism, molecular mechanism of monoculture cropping problem of Rehmannia, identification and exploitation of EST-SSR in our further study.Exploited and identified the EST-SSR of Rehmannia for the first time:on the basis of high-throughput transcriptome library, we exploited1018EST-SSR loci which were equal or greater than18bp, the length statistics indicated:the number of SSR whose length were18bp was the largest, the maximum length was74bp, the number of SSR loci decreased with the length increasing. The type statistics of SSR indicated:the number of dinucleotide was721and was the largest, taking up70.8percent, followed by trinucleotide and six nucleotide, they were486and578respectively. We found AG/GT was the most repeat type (457); secondly, AC/GT (256) and AAG\CCT (137), each number was more than100; and the number of AAAAT/ATTTT was the least. Besides, most of EST-SSR loci were located in CDS regions and UTR regions. Designed the primers of Rehmannia EST-SSR loci (≥20bp) which were selected and identified, and the number of SSR loci which were designed primers successfully and had enough length in their SSR loci flanking regions was320. Based on the transcriptome library, the exploitation of EST-SSR loci laid a solid foundation for further Rehmannia SSR loci polymorphic identification.We established the best optimization system of Rehmannia EST-SSR for the first time:combining with the orthogonal test, we firstly constructed and established the best amplification system of Rehmannia EST-SSR by optimizing the concentration of template DNA, primer, Taq enzyme and dNTP. The optimization results showed: the optimum range of template DNA concentration was80-480ng, the optimum range of primer concentration was0.1-0.9μmol·L-1, the optimum range of Taq enzyme concentration was0.5-1.5U, and the optimum range of dNTP concentration was100-200μmol·L-1. Via Orthogonal optimization of different PCR amplification factors combinations; our study screened the most practical, the most convenient and the most economical Rehmannia EST-SSR amplification system, that was:the total volume was15μL, the template DNA concentration was80ng, the primer concentration was0.1μmol·L-1, the Taq enzyme concentration was1.5U, and the dNTP concentration was150μmol·L-1. The reaction was carried on55℃in30cycles The availability of320Rehmannia EST-SSR primers which were selected and designed in our study was verified, and the result showed:there were180available primers which were amplified expectant bands steadily, it indicated the Rehmannia EST-SSR identified and exploited in this study were reliable.Exploited and established the Rehmannia polymorphic EST-SSR markers library for the first time and classified the cultivated Rehmannia germplasm resources and wild resources according to genetic relationships preliminarily:selected12germplasm resources from36germplasm resources gathered in this study and screened the180pairs of identified primers for polymorphism. The result showed80pairs of primers had higher polymorphism, polymorphism ranged from0.43to0.88, the average polymorphism was0.56. Selected5pairs from polymorphic EST-SSR primers, then appraised and clustered the genetic relationships among36cultivated and wild Rehmannia germplasm resources preliminarily, the result showed36Rehmannia germplasm resources were clustered into3categories:they were wild germplasm, related wild species and cultivated germplasm. Genetic relationships were most closed among the cultivated Rehmannia germplasm resources. Significant clustering of genetic relationships reflected the polymorphic EST-SSR primers identified and selected in this study were reliable sideways. The polymorphic EST-SSR selected in this study provided important molecular basises for genetic relationship identification and cluster analysis of more Rehmannia germplasm resources, and provided important molecular assisted breeding methods for breeding new Rehmannia cultivars which had higher heterosis.Under the circumstances that the genome information of Rehmannia glutinosa was severely lacking and the genetic resources of Rehmannia glutinosa was less, our works was the first time to construct the transcriptome library of Rehmannia glutinosa using Solexa sequencing technology, and optimally assembled the greatest quality and the largest flux transcriptome library sequences in present research on Rehmannia combined with different sequencing platforms. On this bisis, our study constructed and identified the first Rehmannia glutinosa EST-SSR markers using biological method and acquired the polymorphism marker library of Rehmannia glutinosa by using the existing varieties for the first time. Then appraised and clustered the genetic relationships among the existing main cultivated and wild Rehmannia germplasm resources for the first time. The obtaining of these basic works will elaborate the genetic relationships among the germplasm resources of Rehmannia glutinosa varieties breeding in molecular aspects.The cluster analysis of Rehmannia glutinosa varietal germplasm resources made the combining ability testing of different groups of Rehmannia glutinosa varieties breeding possible. Accelerate the application of SSR molecular markers to breed Rehmannia glutinosa new varieties and lay the groundwork for breeding higher heterotic, high-yielding and high quality Rehmannia glutinosa new varieties.