| Nuclear microsatellite (nSSR) and chloroplast microsatellite (cpSSR) are very useful molecular marker tools, widely used in the study of plant genetic structure, phylogeny and relationship. But there are few nSSRs and none cpSSRs of Prunus mume; this greatly limited the application of nSSR and cpSSR in P. mume. Dual-suppression-PCR and Accurate forecast methods were used to isolate nSSR in P. mume. These informational loci will be very useful in the study on the genetic structure, origin, evolution of P. mume and its closely related speciesIt is very close among the relationship stone fruit trees. They can be propagated both asexually and sexually, and easily hybridized naturally and artificially with other stone fruit trees. These features made the genetic background of stone fruit trees rather complex. Previous studies on taxonomy and genetic polymorphism for P. mume were carried out using morphology, cytology, sporule, isoenzyme markers, RAPD, AFLP, SNP and SSR. However, there are still some puzzles. Sequence analysis is a new accurate method with higher efficiency and has been used in the research of phylogeny, genetic structure, introgression in kinds of plant. To settle those puzzles nuclear ITS and chloroplast atpB-rbcL were used to construct the phylogeny of stone fruit trees.1. Prunus mume Sieb. et Zucc, as an ornamental and fruit tree, has been cultivated for centuries in the East Asian, especially in the southern part of China. But much remain unknown about genetic variation and origin of various cultivars. Microsatellite (simple sequence repeat, SSR) is a very prevalent, effective molecular marker. However, there have been few attempts to isolate and characterize SSRs in Prunus mume. To develop more SSRs for the genetic analysis of P. mume, we modified the protocol, called dual-suppression -PCR. A total of eleven SSRs, five (GA)n and six (AC)n, were successful isolated. Eight SSRs were found to be polymorphic in a test sample of 11 P. mume cultivars. The observed number of alleles per locus ranged from 2 to 8 and observed heterozygosity ranged from 0.33 to 1.00 with an average of 0.71, suggesting a high degree of variation in isolated loci. These informational loci will be very useful in the study on the genetic structure, origin, evolution of P. mume and its closely related species.2. At present, many microsatellite-enrichment methods were proposed to increase microsatellite (SSR) isolation efficiency, but it is a complete black-box about the SSR distribution in the insertion fragment in the clone before being sequenced, so the isolation efficiency is still low, which greatly limited the application of SSR. Here we constructed a new model, accurate forecast (AFC). The model can eliminate those useless sequences before being sequenced to gain 100% efficiency and has high feasibility, transferability and fits all the methods with SSRs enrichment (just add it to the end). To simplify the calculation procedure the model was transformed into database software 'Accurate Forecast Microsatellite 1.0'. After modified affinity capture to enrich microsatellite, positive clones were detected by a nested PCR and the data were inputted into the software. The first fifteen anterior clones in the recommendation list created automatically by software were sequenced and all of them sequences were usable for SSR isolation, containing SSR sequences and their corresponding flanking sequences, which were long enough to be used for primer design. Compared with Affinity Capture and Dual-Suppression-PCR, AFC pipeline has higher SSRs enrichment (65%), high sequence percentage (100%) and less cost per SSR locus. It is easy to be operated. It can be a new powerful SSR isolation approach, especially for non-model organisms.3. To comprehend the distribution rule of plant chloroplast microsatellites (cpSSRs) and develop the universal cpSSR primers for stone fruit trees, the complete genome of Arabidopsis thaliana Chloroplast was downloaded and analysed. The mononucleotide cpSSRs were preponderant (78.6%), the dinucleotide cpSSRs, the trinucleotide cpSSRs and the others cpSSR accounted for 19%, 2.4 % and 0%, correspondly. The (T)n and (A)n cpSSRs were the most type in the mononucleotide cpSSRs (98.5%) while all the dinucleotide cpSSRs were (AT)n. 41 pure SSRs (62.1%), 23 interrupted SSRs (34.8%) and 2 multiple cpSSRs (3.1%) existed in the the mononucleotide cpSSRs.4. Two methods were used to develop the universal cpSSR for stone fruit trees. 1) Non-coding region sequencing (NRS). Total Genomic DNA of Prunus mume 'Koshu Koume' was extracted. A nested PCR was performed to amplify and screened 16 chloroplast intergene sequences (IGSs) and 1 intron. 8 IGSs were successfully amplified in the secondary nested PCR. After sequencing 9 mononucleotide cpSSR loci, (A)n or (T)n with n≥8, were found. 2) Bio-informatics method. 33 chloroplast sequences of peach, plum, mume, apricot, cherry were downloaded form the Genbank. 38 mononucleotide cpSSRs, (A)n or (T)n with n≥8, were obtained by directly search, 8 of them with n≥10 were choosed to design universal primer. 6 pairs of cpSSR primer developed using NRS were tested in 24 Prunus genotypes and all of them were polymorphic. 18 fragments were yielded and formed 14 haplotypes. The high Heterozygosity (0.96) indicated there were rich haplotype types in stone fruit trees. But apricot and mume had lowest Heterozygosity (0.32) and only 2 haplotypes with disequilibrium distribution. This showed that apricot and mume had simplex origin and limited genetic background. These new developed cpSSRs provide a new alternative efficient tool to analyze the origin and evolution of stone fruit trees.5. To reconstruct phylogeny of stone fruit trees, the atpB-rbcL noncoding spacer of chloroplast DNA from 24 genotypes of peach, plum, apricot, mume and cherry, 4-5 genotypes per specie, were sequenced and analyzed. With Zippeliana as outgroup, the data set was tested with 56 evolution models with PAUP and the score was used to screen the best model and parameter with Modeltest. Genetic distances, variances were calculated and the maximum parsimony tree was constructed with Mega. The result indicated: 1. Molecular evolution rates were different among stone fruit trees and the distribution of variance was unbalanced; 2. An initial split from the backbone of the Phylogenetic tree indicated that cherry was more original than other species. Mume had closest relationship with apricot and closer with plum. As a monophyletic group, stone fruit trees were come from an original plant with two clades, one evolved to cherry and the other to other stone fruit trees.6. To analyze the molecular evolution and phylogeny of stone fruit trees, the internal transcribed spacers (ITS) of nuclear ribosomal DNA from 16 genotypes of peach, plum, apricot, mume, and cherry, 2-4 genotypes per species, were sequenced and analyzed with 6 ITS sequences downloaded from the GenBank. Prinsepia sinensis as outgroup, the maximum parsimony tree was constructed with Mega. An initial split from the backbone of the phylogenetic tree indicated that cherry was more original than other species. Because Prinsepia sinensis was greatly different with others, the second way of rooting with cherry was performed. The broad data set was tested with 56 evolution models with PAUP and the score was used to screen the best model and parameter with Modeltest. Mega was used to calculate genetic distance, variance and construct maximum parsimony phylogenetic tree. The result indicated: 1. Molecular evolution rate and information were different between ITS1 and ITS2, and also among stone fruit trees; 2. The evolution order of 5 stone fruit trees was analyzed. Cherry, plum and apricot were come from an original plant, and then plum evolved to peach and apricot to mume; 3. Prunus mandshurica is more original than P. sibirica and P. armeniaca. The evolution order of peach is from P. andersonii to P. davidiana and then to P. persica (P. ferganensis); 4. It is reasonable that stone fruit trees are divided into 4 subgenera. |
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