Development Of SSR Molecular Markers Based On The Transcriptome Data Of Gerbera Jamesonii Bolus.

Gerbera jamesonii Bolus.is a kind of cut flowers with high ornamental value and practical value.Gerbera germplasm resources are very rich and diverse.Up to now,however,no effective calssification method has been established.Therefore,it is very important to study the effective classification of the existing gerbera varieties and the genetic diversity.In this paper,the morphological traits were first analyzed.Then based on thetransgenic data of gerbera,the EST-SSR markers for gerbera were developdand core sites and the validation of ISSR markers was studied.The results are as follows:1 Genetic diversity analysis of gerbera germplasm resources based on morphological markersIn this study,seven quantitative traits and 16 morphological traits of gerbera were analyzed by using Gerbera DUS test method,and was the used for the genetic diversity analysis.The results showed that the coefficient of variation of 7 quantitative traits ranged from 10.4%to 28.3%with an average value of 17.4%.The coefficient of variation was in the order of flower diameter,leaf width,pedicel length,leaf length,Petal length and inflorescence diameter,among which the variation of pedicel length is the largest and the variation of flap width was the lowest.A total of 49 alleles were detected in 47 morphological traits.The average number of alleles per locus was about 3,ranging from 2 to 6,and 16 traits Polymorphism allele variability,accounting for 100%of the total number of traits;the average number of effective alleles was 1.4222,the amplitude ranged from 1.1978 to 1.7068;Nei’s genetic distance index was 0.2759 in average,the amplitude was 0.1557-0.4135;Shannon’s diversity index was 0.4373 in average,the variation was 0.2819～0.6038.Flower color and the number of color types on the outer surface of the tongue was significantly higher than that of other traits.Therefore,these two traits could be used as index for calssification.The results showed that the similarity coefficients of the morphological traits were 0.57～0.96.When the similarity coefficient is 0.62,47 gerbera test varieties can be divided into three categories.According to the classification results and the diversity index of morphological traits,four traits,i.e.he color of flowers,the type of petals,the length of pedicels and the number of flowers on the outer tongue,were found to have a good effect on the classification of Gerbera.2EST-SSR loci characterisitics analysis of gerberaThe results showed that the frequency of SSRs was 14.15%.Totally,18044 SSR loci were retrieved and the frequency of SSRs was 16.69%.There were 2291 unigenes with more than one SSR locus;999 were found in the complex SSR locus with a frequency of 0.92%.From the frequency of EST-SSRs in gerbera,the dinucleotide and trinucleotide motifs were predominantly nucleotides(98.1%),which were 57.17%and 40.93%.Fifteenteen repetitive motifs were retrieved from the number of repeated motifs.Among them,the nucleotide sequences of the three nucleotide sequences and the four nucleotide sequences were the largest,60 and 50 respectively,and the proportion of the three kinds of motifs was 39.47%and 32.89%respectively,all of which belonged to all EST-SSRs And 72.36%of the motifs.From the point of the repetitive motif,the number of GA primers in the dinucleotide motif was 506 and was the most repetitive motifs.The AG primers and AC primers ranked the second,respectively(AG)n=(CT)n total of 1665,the frequency of 1.54%,the proportion of SSRs was 28.24(P<0.05),and the number of CG primers was at least 13 times.(AAT)n=(ATT)n is the optimal repetitive motif type,frequency of appearance was 0.56%and the proportion of SSRs was 10.31%.The number of AAT motifs is the highest,a total of 148 times.(ACT)n=(AGT)n,the proportion of SSRs was only 0.59%.The results showed that the EST-SSR loci were rich in the genome and the genome motifs quantity were abundant.3 Establishment and Optimization of EST-SSR Reaction System of gerberaIn this experiment,the optimal reaction system of EST-SSR was determined by L25(55)orthogonal test.The concentration of DNA template was 15 ng/μL,the concentration of primer was 0.8 mmol·L-1,the concentration of Mg2+ was 2.4 mmol·L-1,dNTPs at a concentration of 0.25 mmol·L-1,Taq enzyme concentration of 2.0 U.Non-optimized test factor ddH2O was complemented to 25 μL.4 The research of gerbera EST-SSR markersAfter rescreening,11 SSR-labeled primers were successfully used to amplify the target fragment of 50 gerbera test materials,and the polymorphism was higher,specificity and reporducibity were better.The harvest rate of EST-SSR marker was 22%.The results showed that the EST-SSR primers were able to amplify the product in different cultivars.The core locus sequence and the flanking conserved region were detected in the bands.The results showed that the EST-SSR markers had good reproducibility,high accuracy and high reliability.The results showed that the EST-SSR markers were highly reproducible,and the accuracy was strong.A total of 201 bands were obtained from 50 copies of gerbera markers,of which 195 were polymorphic bands,17.72 polymorphic bands were developed per pair of primers,the average polymorphism ratio up to 97.01%.To sum up:the gerbera EST-SSR marker is highly reproducible and has abundant polymorphism.5 The results of EST-SSR marker clustering showed a certain correlation with some morphological traits11 pairs of Gerbera SSR marker primers can be used to classify 50 parts of Gerbera test material at DNA level.The genetic similarity coefficient of 50 copies of Gerbera test material is between 0.63 and 0.98.When the genetic similarity coefficient is 0.701,50,the varieties of Gerbera can be broadly divided into three categories.The results of morphological studies on the morphological characters of Gerbera were found to be based on the clustering results of the EST-SSR markers at the molecular level and the size of the gerbera leaves,the number of the upper surface of the outer tongue-shaped flowers was 2,and the petal type had the same morphological traits.And the correlation with the morphological traits such as the degree of bubble bulging,the top traits of leaves,the depth of fissure at 1/3 of leaf and the degree of green on top of leaf were lower.6 Comparative Analysis of EST-SSR Markers and ISSRFrom the amplification effect,155 amplification sites were obtained by using 16 ISSR markers.The number of polymorphic bands was 145 and the polymorphism ratio was 93.55%.The polymorphism of EST-SSR marker and ISSR was higher than that of ISSR,and the number of amplified bands and polymorphic loci was significantly higher than that of ISSR,Showing a greater range of amplification.The two methods of labeling were divided into three groups,which were divided into four subclasses in the second category.Although each group contained different varieties of gerbera,the quantity was different.The results showed that the coincidence rate of the two markers was 70%.The results showed that the EST-SSR markers of gerbera had a certain reliability and stability in the research of kinship analysis.Based on the study of morphological traits,sequencing of amplification results and validation of IS SR molecular markers,it can be seen that the development of EST-SSR markers of gerbera can be used in the research of classification and genetic relationship analysis of Gerbera species.