Changes Of Phenotype And Gene Expression In Early Generations Of The Synthesized Allotetraploid Cucumis×hytivus

Polyploidization is a prominent process and has played an important role in the evolutionary history of higher plants. Recent studies indicated that extensive changes of genome composition and gene expression occurred in the early stage of genome formation of allopolyploid, also, novel, non-Mendelian phenotypes arised in synthesized allopolyploids. These changes have played a significant role in the stabilization of newly formed species'genome. The development of molecular markers and comparative genomics have provide important basis for understanding of allopolyploidization. Studies on such changes of nature allopolyploids were compared to their candidate diploid progenitors. While nature allopolyploids formed thousands upon thousands years ago, their genomes have evolved for a long time, and diploid progenitors also have evolved or not existed. So it is difficult to expore the concrete information during allopolyploid evolution.Newly synthesized allopolyploids and'young'allopolyploids can be a very useful model system to study allopolyploidization for their clear genetic relationship, and the information at the early stage can be obtained exactly. With the newly formed allopolyploids, the changes of phenotype and gene expression can be studied accurately compared to their diploid parents, which might help enriching the evolutionary theory of allopolyploidization. In this work, the characterization of changes of morphological traits, cytology behavior and gene expression in early generations of C.×hytivus were studied, and expression and sequence variation of cucumber por gene in C.×hytivus were also investigated. Some mechanisms resolved in these changes were discussed. The detailed results are as follows:1. Changes of phenotype in early generations of the synthesized allotetraploid C.×hytivus The characterization of changes of morphological traits, meiotic chromosome behavior and pollen fertility in early generations (S1-S4) of the synthesized allotetraploid C.×hytivus were studied. The results from observation of morphological traits indicated that these traits showed instability, and some traits including internodes'length, lateral branches, leaf thickness, pedicle length of male flower and ovary length showed high coefficient of variation; From S1 generation to S4 generation, the leaf thickness got to thinner, the ovary tended to shorter and the appearance of first female flower became later, while other traits showed variation without regularity.Investigations on meiotic chromosome behavior showed that most meiotic chromosome configurations at metaphaseⅠphase were bivalents. The frequency of bivalent at metaphaseⅠincreased, from S1 generation to S4 generation, while which of univalent and multivalent decreased. The number of cells with nineteen bivalents also increased obviously; at metaphaseⅡ, the allotetraploid mainly produced tetrads, and the frequency of tetrads increased from S1 generation to S4 generation. Accordingly, pollen fertility was recovered generally. Above results indicated instability of morphological traits and diploidization of meiotic chromosome behavior in the evolution of early generations of the allotetraploid C.×hytivus.2. Changes of gene expression in early generations of the synthesized allotetraploid C.×hytivusChanges of gene expression played an important role in the evolution of plant allopolyploids. Characterization, including frequency, time and type of the changes of gene expression between the first two self-pollinated generations (S1 and S2) of the synthesized allotetraploid C.×hytivus and in its diploid parents was analyzed with cDNA-AFLP and reverse-Northern blot technique. Sequences similarity of genes involved in changed expression were also analyzed with BLAST package. The results from cDNA-AFLP analysis showed that 36 (3.37%) genes showed silencing (27) or activation (9) in allotetraploids. These changes initiated in S1 or S2 generation. The silenced/activated genes included rRNA and protein-coding genes. Further reverse-Northern blot analysis validated the above results. Thus four types of changes of gene expression were observed, including silencing of genes from both parents, maternal parent, paternal parent, and gene's novel expression. Above results indicated rapid gene silencing and activation in early generations of C.×hytivus, which contributed to the evolution of the allotetraploid. Changes of expression level of homoeologous rpl2 gene from diploid parents were analyzed in early generations of allotetraploid C. x hytivus with cDNA-SSCP technique. The results indicated that homoeologous rpl2 genes showed different expression level in C. X hytivus. The expression level of gene from C. sativus was high than that from wide species C. hystrix, and it also showed instability in early generations. Above results indicated that allopolyploidy induced changes of expression level of homoeologous gene in allotetraploid C. x hytivus.3. Expression and sequence variation of cucumber por gene in the newly synthesized allotetraploid C.×hytivusThe phylogeny of the plant por gene was investigated. Identities of cDNA sequence of Por gene and amino acid sequence of POR in some plant species were analyzed by BLAST, and gene tree was also constructed with MEGA software version 4.0. The results showed that cDNA and protein sequences were conservative in plant species, which indicating the common function of these genes. However, there also existed sequence difference of various por genes, and sometimes, identities of cDNA sequence of por genes from various species were higher than that from one species.The molecular evolution of the cucumber por gene in early generations of the synthesized allotetraploid C. x hytivus was investigated. The results from gene expression analysis by RT-PCR showed that the cucumber por gene was silenced in the S1 generation, and re-activated in the S2 generation. In the S3 and S4 generations, the transcripts remained activated but sequence changes were observed. Further analysis indicated that base substitutions, including two transitions and one transversion, occurred in the S1 and S3 generation respectively, and in the S3 generation, an intron was found to be retained in the transcript. This indicates allopolyploidy induced rapid silencing and mutation of the cucumber por gene. Further, gene mutations such as base substitution and intron retention are also modes of evolution for duplicated genes in newly formed polyploid.