| Agropyron mongolicum Keng,is a perennial forage belonging to Triticeae in Gramineae family.It is diploid(with PP chromosome,2n=14).It is native to the Desertification Grassland in Northwest China.It has the characteristics of early turning green,long green period,soft stems and leaves,high nutrient content and good palatability.It also has the functions of windbreak and sand fixation,soil and water conservation and desertification grassland improvement.As a wild related species of wheat crops and rich in excellent resistance genes,it has important breeding value.However,due to the late start of the research,the information on transcriptome,molecular markers and germplasm identification of A mongolicum is very limited,which seriously limits the development of A mongolicum germplasm resources.In this study,we collected 11 different tissues from the roots,leaves,seeds and seedlings of A mongolicum,extracted the total RNA,and then mixed the total RNA to build the library.Then,we used Illumina hiseq2000 sequencing platform for transcriptome sequencing.According to the sequencing results,we developed 10 pairs of polymorphic EST-SSR molecular markers of A mongolicum,and detected the universality of the newly developed EST-SSR primers in Agropyron species,These polymorphic molecular markers were used to construct DNA fingerprints of Agropyron species.The main results are as follows1.A total of 6.85G of transcriptome was obtained from 11 different tissues.By assembling the sequencing data,56,684 functional genes were obtained.Comparing these functional genes with Nr,Nt,KO,Swiss-Prot,Pfam,GO and KOG databases,the number and proportion of annotation were 38,231(67.44%),42,470(74.92%),13,500(23.81%),26,638(46.99%),26,608(46.94%),26,608(46.94%)and 10,272(18.12%)respectively.All functional genes were successfully annotated.All Unigene were predicted by CDS,and a total of 58,524 coding sequences were predicted,of which 30,531 were from the comparison of Nr and Swiss-Prot protein library,and 27,993 were from estscan prediction.2.Using MISA software to identify 56,684 SSR loci of functional genes,a total of 7,880 functional genes were identified,including EST-SSR loci.A total of 8,382 pairs of primers were designed using primer3.0,and 45 pairs of primers were randomly synthesized for subsequent research.By changing the ratio of specific forward primer and universal M13 primer,the three primer PCR system was optimized to improve the accuracy of subsequent data analysis.The results showed that 10 pairs of primers showed high polymorphism.The average percentage of polymorphic bands was 57.93%.The results showed that 9 pairs of EST-SSR primers showed high polymorphism,the average percentage of polymorphic bands was 78.43%.3.Based on the number of polymorphic bands and the difficulty of statistical analysis of amplified bands between 10 pairs of primers and Agropyron germplasm,primer 4 and primer 23 were finally selected to identify 5 A mongolicum materials;Primer 4 and primer 21 were used to identify 6 Agropyron materials.These three pairs of primers can distinguish 11 Agropyron materials simply and efficiently,andcan be used for germplasm identification.In this study,a transcriptome database covering almost the whole growth period of A mongolicum was established,which enriched the transcriptome information of A mongolicum.By mining the transcriptome information,a number of polymorphic EST-SSR molecular markers were developed,and the DNA fingerprints of 11 Agropyron germplasm were constructed by using the developed polymorphic primers.Fluorescence capillary electrophoresis was introduced into the study of molecular markers of A mongolicum,and the three primer PCR system of A mongolicum was successfully optimized,which laid an experimental foundation for the follow-up automatic and large-scale exploration of A mongolicum germplasm resources. |
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