| Mei (Prunus mume) originated in China and belongs to the sub-family Prunus within the Rosaceae family and is widely cultivated in East Asian countries. Mei has been used for7,000years and the cultivation history for over3,000. With the rapid development of biotechnology, it has reported that understanding of the growth and development of Prunus mume from the level of gene were gradually received some attention, as well as the genetic evolution of the stone fruit plants. In recent years, molecular markers have been widely used in plant taxonomy and phylogenetic analysis. And there are rich germplasm resource in China, the studies of classification and phylogenetic relationship of Prunus germplasm resources had conducive to the collection, preservation, identification innovation and rational utilization of Prunus mume germplasm resources. In this study, the cDNA library of the mei flower and fruit was constructed. Combined with part of EST sequences from other stone fruit plants, the differentially expressed genes involved in fruit development between Prunus mume and Prunus armeniaca were analyzed on the level of the transcriptome by using microarray experiments. In addition, the isolation mechanism between Prunus mume and Prunus armeniaca was also discussed in this study. The main results are as follows:1. The first cDNA library of Prunus mume derived from the RNA isolated from the flower and fruit at different developmental stages was constructed with Creator SMART cDNA Construction Kit. Analysis indicated that the cDNA library had a high titer of1.4×106pfu/mL, with a recombinant efficiency about97%. The insert sizes of the library were1-3kb based on a PCR analysis of30randomly chosen clones, suggesting that the cDNA library of Prunus mume flower and fruit has been successfully constructed and could serve well for the cloning and functional analysis of important genes involved in the development of flower and fruit.2. In this study, we constructed a cDNA library of Prunus mume flower and fruit, sequenced10,123clones of the library, and obtained8,656expressed sequence tag (EST) sequences with high quality. The ESTs were assembled into4,473unigenes composed of1,492contigs and2,981singletons and that have been deposited in NCBI (accession IDs: GW868575-GW873047), among which1,294unique ESTs were with known or putative functions.3. In this study, from10,123Prunus mume ESTs screened using MIS A software, and a total of1233putative simple sequence repeats (SSRs) in the P. mume unigene dataset were mined from935ESTs with frequency of12.18SSR loci distributed over100ESTs. Di-and tri nucleotide repeat EST-SSRs were dominant, accounting for35.52%, and41.36%, respectively. Furthermore, we randomly tested42pairs of PCR primers flanking potential SSRs, with24primer pairs showing amplifications while17were polymorphic, where14pairs were identified and73.08%of the PCR amplification bands as true-to-type SSR loci. We further used the14EST-SSR primer pairs to test the transferability on other Prunus species, such as peach, apricot and plum. The result showed that100%and89%of the primer pairs produced target PCR bands in the apricot and two other species, respectively. A high level of marker polymorphism was observed in the apricot species (89.6%), lower in the plum (65%) and peach (46%), and the clustering analysis of the three species.4. Differentially expressed transcripts along the entire fruit life cycle base on two P. mume cultivar’Xiyeqing’,’Ruantiaohongemi’and two P. armeniaca’Manao’,’Zhenkui’ were first identified, by using microarrays spotted with10,641ESTs collected from P. mume and other Prunus EST sequences. A total of1,418ESTs were selected after quality control of microarray spots and analyzed for differential gene expression patterns during fruit development of P. mume and P. armeniaca. Among them,707up-regulated and711down-regulated differentially expressed genes showing more than2.0-fold differences in expression level were annotated by GO based on biological processes, molecular functions and cellular components. And54%(766) genes were found involved in17biological processes such as metabolic process, cellular process, response to stimulus, developmental process, multi-cellular organism process, localization, biological regulation, reproduction, signaling. The KEGG results also showed that those genes encoding enzymes were involved in several pathways primarily focused on Carbohydrate Metabolism including Glycolysis/Gluconeogenesis, Citrate cycle (TCA cycle), Galactose metabolism, Starch and sucrose metabolism, and Biosynthesis of Other Secondary Metabolites containing Phenylpropanoid, Flavonoid, Carotenoid, Hormone and lignin biosynthesis. qRT-PCR expression patterns of12(75%) of these genes were in agreement with their microarray analysis results, and it should be noted that all the selected genes were detected with different expression during the stages of fruit development.5. In this research, multiple alignments were performed between the unigenes of Prunus mume and both EST database of P. armeniaca and P. Persica published in GenBank,592homologous sequences were found with a total length of235576bp, and the average length and homology were437bp and97.5%, respectively. The Blast results also showed that340of them had the corresponding functional annotation,183were unknown proteins, and the remaining69had new gene sequence information. The amount and frequency of nucleotides were further analyzed, where8,818SNPs were found having a total frequency of26bp per SNP. The amount of SNPs compared in pairs was significantly less than the number among the homologous sequences. In addition, the cluster analysis result by using the obtained SNP information showed that the relationship between Prunus mume and P. armeniaca was closer, and they were distantly related with P. persica,6. Genetic relationships among68mei cultivars(Prunus mume Sieb. et Zucc.), including36flowering mei and32fruiting mei cultivars, were assessed using single nucleotide polymorphism (SNP) markers. The sequence alignments of nine group genomic sequences consisting of a total length of2,916bp, amplified from68DNA samples using nine pairs of PCR primers, yielded92SNPs with a distribution frequency of one SNP per32bp. Among these SNPs,51(55.4%) were transitions,34(37%) were transversions,4(4.4%) were InDels and3(3.3%) were others. Of the92SNP loci in these68mei cultivars, there are several different existing-states of them among different cultivars:13(14.1%) were shown to be two kinds of homozygotes in different cultivars;22(23.9%) were recognised as heterozygotes in some cultivars and two kinds of homozygotes in other cultivars;50(54.3%) were heterozygotes and only one kind of homozygote in other cultivars. Cluster analysis of the68mei cultivars was carried out using the92SNPs, and a consensus cladogram was constructed. All the mei cultivars could be classified into11groups, instead of two groups consisting of only flowering mei or fruiting mei, respectively. This suggested that flowering and fruiting mei trees are genetically similar differently in their floral and fruit characteristics.7. In this study, the divergent flowering phonologies and geographic distribution were reported that they were in substantial reproductive isolation between two important species of Prunus armeniaca and Prunus mume that can be cross-compatible. The investigation results show that geographical isolation and their flowering phenology isolation were important barrier in crucial mode of pre-zygotic isolation to maintain species boundaries of P. armeniaca and P. mume. The molecular evidence for pre-zygotic isolation between P. armeniaca and P. mume are also presented. Ecological and molecular factors provide adequate proof that P. armeniaca and P. mume could be two independent species that are however genetically related. Conclusions:These comparative analyses demonstrate that pre-zygotic isolation plays an important role in evolution between P. armeniaca and P. mume and shows that’Xingmei’is the sterile hybrid generated by hybridization of these two species. The empirical and molecular evidences in this paper can be informative and helpful for the further studies on evolution and speciation of P. armeniaca and P. mume and even for the entire Prunus genus. |
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