| Cornus plants belong to the Cornacease,and most the species are landscape trees of high ornamental value,C.flordia,C.kousa and C.nuttallii have attracted wide attention because of their unique bract characteristics,and C.flordia is a state flower in Missouri,north Carolina,Washington state and other places because of its high ornamental value,which has great development and utilization value.At present,the genetic background of Cornus germplasm resources in the domestic market is not clear,and the variety names are confused.In this study,19 species and varieties of Cornus were used as test materials.The SSR molecular markers of Cornus were developed by transcriptome sequencing,and the SSR-PCR reaction system was optimized.The phylogenetic relationship and the fingerprints of some varieties were constructed to provide a basis for better utilization of Cornus flower germplasm resources.The main research results are as follows:(1)The RNA-seq technology based on Illumina platform was used to sequence the transcriptome of C.florida,and rich transcriptome data was obtained.The bioinformatics software was used to excavate SSR sites of Unigene sequences obtained by transcriptome sequencing.A total of 30401 SSR sites were obtained.The frequency of SSR distribution was 22.90%and the average distribution distance was 2.88kb.The repeat types of SSR loci are mainly single nucleotide repeats and dinucleotide repeats,which account for 45.73%and 37.98%of all loci,respectively.In general,the number and types of SSR loci in the transcriptome of C.florida are relatively abundant,the distribution density is large,and there is potential polymorphism.(2)Orthogonal design experiment was used to optimize the SSR-PCR reaction system of Cornus,and an SSR-PCR amplification system suitable for Cornus was established.The optimal reaction system was:under the total reaction system of 25?L,the optimal conditions are a primer concentration of 0.4?mol·L-1,a template DNA content of 80 ng,and a Premix Taq enzyme(TaKaRa)content of 12.5 ?L.The most important factor in the reaction system is the content of Premix Taq enzyme.(3)Through the development of primers,31 of the 50 designed primers were able to effectively amplify with an amplification efficiency of 61%,of which 18 primers were polymorphic,accounting for 58.06%of the amplifiable primers.A total of 131 polymorphic fragments were detected in 18 pairs of primers,and 3 to 12 alleles could be detected at each SSR locus.The average number of alleles observed per locus was 7.278,indicating that these 18 primers it can show good polymorphism in test materials.(4)The above primers were used to perform UPGMA cluster analysis on 19 Cornus germplasm resources,and the genetic similarity coefficients of various germplasm resources were calculated to be in the range of 0.6183 to 0.9160.The genetic similarity coefficient is at the level of 0.67,and 19 materials can be divided into two categories:Type ? includes all North American Cornus germplasm except the cultivar 'North Star',and Type ? includes the cultivar 'North Star' and all East Asian Cornus germplasm The clustering results basically accord with the characteristics of traditional taxonomy,which can reflect the genetic relationship between germplasm(5)Using 18 pairs of primers to PCR amplify 11 Cornus varieties,and use the primer combination method to construct fingerprints of Cornus varieties,which can completely distinguish 11 Cornus varieties,and provide a basis for variety identification. |
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