| Eucommia ulmoides Oliver is a dioecious perennial woody plant with high medicinal and economic value. The utility value exist difference between male and female of E. ulmoides, especially the gutta-percha and other secondary metabolites. Also, it was difficult to identify sex by morphological or cytological methods before flowering, and it's need over 7 to bloom. In order to enhance the effective conservation and sustainable utilization for E. ulmoides Oliver germplasm resources, we need to reseach genetic background. Therefore, we based on the female and male transcriptome datum of E. ulmoides to develop SSR molecular markers, which could be used to research functional gene mining, molecular marker-assisted breeding, genetic diversity analysis, genetic map construction, etc. This study based on the transcriptome of dioecy E. ulmoides to develop SSR molecular markers.The results showed were as follow: 1 SSR Minging and characteristics analysis in E. ulmoides dioecy transcriptome databaseSSRs were mined and its characterization were analyzed by MISA software based on the unigenes of the female and male E. ulmoides transcriptome datum, we obtained 74 230 SSRs that distributed in 61 629 unigenes, with the frequency was 33.99%. Among of these SSRs, the most repeat motifs with length of 1224 bp were discowered, and the most repeat numbers of the motif were 11 to 15. Mononucleotides, dinucleotides and trinucleotidesappeared to be the most abundant repeated motifs, accounted for 98.6% in total SSRs, in which A/T?73.16%??AG/CT?10.91%??AAG/CTT?1.14%?were the most abundant respectively.2 Optimization of SSR-PCR reaction system for E. ulmoidesAccording to the orthogonal test of L9?34?, the optimized amplificationswere performed in 20?L volume containing 10 ?L of 2 × Premix Taq DNA polymerase(0.05 ?mol·min-1·?L-1), 1 ?L of DNA(25ng·?L-1), 0.5 ?L(10?mol·L-1) of each primers, 8 ?L ddH2 O. 3 Primer selection and polymorphism detectionOne DNA sample of E. ulmoides was used to detect the amplification effect and optimize annealing temperature of 210 primers that were randomly selected, one hundred and forty pairs of primers in 210 primers?66.66%? were successful amplificated, and then we used 10 DNA samples from 9 different regions to detected the efficiency of SSR primers, obtained 15 stable, repeatable polymorphic SSR loci and 57 alleles were observed with 3.8 alleles in one locus on average; observed heterozygosity?Ho?, expected heterozygosity?He? and the polymorphism information content valued?PIC? range from 0 to 1.00, 0.28 to 0.78 and 0.26 to 0.70, respectively, and the mean value of PIC was 0.44. All of the 15 SSRs loci had expected repeats motif sequences by sequencing indicated. 4 Genetic Diversity and Phylogenetic Relationship of Cultivated Eucommia ulmoides Olive in Guizhou by SSRIn this study, we used 15 SSRs molecular markers to investigate the hereditary constitution and genetic relationship for the cultivated populations of E.ulmoides in Guizhou. The total of 76 alleles were detected based on 12 populations included 295 samples, the alleles per locus rang from 3 to 10, and average number was 5.067; the average number of alleles per population?na?, expected heterozygosity?He?, and Shannon's index?I? were 4.011?0.533, and 0.996, respectively. And except the percentage of polymorphic loci?PPL? of LL, QNY were 93.3%, the other 10 populations' PPL were 100%, indicated a higher level of genetic variation of E.ulmoides in Guizhou. Only the negative fixation index?F? exist in MT and LL, others were positive value, which implied heterozygote deficiency commonly existed in most populations,because of inbreeding within population. The genetic differentiation coefficient?FST? between populations were 0.0660, suggested genetic variation maily distributed within populations. The UPGMA cluster analysis indicated that grouping wasn't according to the religion location to cluster, it may be the result of artificial seletion in the process of cultivation, QNY had the most genetic distance with other 11 populations. However, genetic distance between XY and RH was the closest. Some suggestions are recommended for both genetic conservation and germplasm collection of E. ulmoides. 5 Development specific male marker in E. ulmoidesWe screened primers by using DNA samples of 6 male and 6 female plants from 7 different regions, which designed primers based on the SSRs locus flanking sequences of dioecious transcriptome library of E.ulmoides.We obtained gender different produces by PCR which has been cloned and analysed, then another 12 male and 12 female plants by used to test reliability of gender marker. The result showed that only one pair primer?EST-Eu059? in a total of 140 pairs EST-SSR primers had different bands between male and female, a common band was about 160 bp?named as Eu-f160 and Eu-m160? in dioecious plant, and then there was an extra 120 bp approximately?named as EuMSM? bands in all male. Sequence analysis indicated that Eu-f160 and Eu-m160 were 156 bp, with the repeat unit?GT?6, and only 1 base was different, that was, the third G base in the female repeat unit became A; and EuMSM was 112 bp without?GT?6. By Blastn comparison, EuMSM was not homologous with the sequences in the GenBank database. We confirmed that there was a male-specific band in 12 female and male plants. |
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