Cytological Analysis Of The Role Of SPO11-1 On Meiosis In Autopolyploid Arabidopsis Thaliana

Polyploidization events occur frequently during plant evolution.The most popular estimate of the proportion of polyploids in angiosperms is 70%.Polyploidization has a meaningful role in plant evolutionary history,and is a driving force for evolution and variation in plants,and is a pathway for plants to maintain genetic diversification and genomic stability.Meiosis is one of the major events during sexual reproduction,allowing organism to switch from a diploid to a haploid.The crucial biological events include homologous chromosome synapsis,recombination and crossover formation.These events allow the requisite segregation of homologous chromosomes.Polyploid has more than two sets of chromosomes;the process of homologous chromosomes recombination in autopolyploid is more complicated.So there is great significance to investigate meiosis related genes in polyploid.In this study,we use the technology of cytogenetic to investigate the function of SPO11-1 during meiosis homologous recombination in autopolyploid Arabidopsis thaliana.Here are our results:1.We select special part of CDs of SPO11-1 sequence,and construct RNAi vector.2.We transform the vector into autotetraploid Arabidopsis using floral dip and drop-by-drop method.We put the seeds on solid medium containing 50mg/L kanamycin to select transgenic plants.The transgenic plants can expand 4 true leaves while the non-transgenic plants turn yellow and stop grow.We verify the transgenic plants by PCR and real-time PCR later.The real-time PCR results show that the expression of SPO11-1 of transgenic plants is obviously falling down.3.Both the diploid and autotetraploid transgenic plants show no apparent different phenotype during vegetative phase.The results show that both the diploid and autotetraploid transgenic plants grow with a similar rate and develop a similarnumber of rosette and cauline leaves and a similar biomass as their wild type neighboring plants.Rosette leaves appear normal and bolting is not delayed.4.Both the transgenic plants show sever defects in reproductive development and considerably reduced fertility.1)They produce much-shortened siliques compared with wild type.We open the mature green siliques in wide-type and transgenic plants.Most of the seeds produced by wild-type diploid are normal(93.6%),with limited evidence of aborted ovules.Whereas only 51.8% of the ovules of the diploid transgenic plants are normal.Most of the seeds produced by autotetraploid wild-type are normal(73%).Conversely,in autotetraploid transgenic plants,more than half of the ovules are aborted,leading to a residual level of seed set.2)We stain the pollen grains with Alexander's solution.The pollen grains produced by wild-type flowers are stained with red color and are similar in size.They have regular shape,the majority of them are round.However,the transgenic plants produce pollen grains that are variable in size and usually smaller than wild-type ones.Some of them are shrink and shriveled.It is indicated that some of the pollens are non viable.The rate of abnormal pollen of wild type diploid is 2.9%,the rate of abnormal pollen of transgenic diploid plants is 52.2%.The rate of abnormal pollen of wild type autotetraploid is 10%,the rate of abnormal pollen of transgenic autotetraploid plants is 45%.We stain the anther using Alexander's solution,too.The amount of pollen grain of transgenic plants is also less than wild type.5.Cytological analyses are conducted to detect the defects during meiosis in the pollen mother cells of transgenic plants.In wild type Arabidopsis,at the onset of meiosis,the indistinguishable homologous chromosomes(chromatins)gradually condense.Paring and synapsis of homologous chromosomes can be seen as synaptonemal complex at pachytene.Chromosomes further condense and become distinguishable with bright DAPI-stained centromeres after completing condensation at diakinesis.At metaphase?,chromosomes behave as quadrivalents or bivalents with several entangled chiasmata.At the transition from metaphase?to anaphase?,homologous chromosomes separate equally in opposite directions.From metaphase?to anaphase?,coherent sister chromatids separate and are pulled apart gradually by the centromere-associated spindles.In contrast,in both diploid and autotetraploid transgenic plants,there are sever defects during meiosis.At leptotene,the behavior of chromosomes appears normal.Paring and synapsis are strongly impaired.The homologous chromosomes can not fully synapsis at pachytene.At metaphase?and metaphase?,some of the chromosomes form univalent instead of bivalents.Some of the chromosomes are away from the equatorial plate.Such univalents are often then retained behind as lagged chromosomes at the first cell division.The lagged chromosomes appear at metaphase?.In telophase?,the chromosomes mis-segregation produces unbalanced cells.The abnormal chromosome behavior rate of diploid wide type at diakinesis,metaphase?,anaphase?,anaphase?is 0%.The abnormal chromosome behavior rate of diploid transgenic plants at diakinesis,metaphase?,anaphase?,anaphase ? is 66.7%,50%,33.3%,27.3%,respectively.The abnormal chromosome behavior rate of autptetraploid wide type at diakinesis,metaphase?,anaphase?,anaphase ? is 75%,16.7%,0%,0%,respectively.The abnormal chromosome behavior rate of autotetraploid transgenic plants at diakinesis,metaphase?,anaphase?,anaphase?is 84.2%,33.3%,33.3%,28.6%,respectively.