Study On Imprinting And Expression Of Endosperm Development Genes In Hybrid Offspring From The Cross Of 2N And 4N Rice

Genomic imprinting is a phenomenon that the differential expression of alleles of the same gene depending on its parental origin in the offsprings of mammals and flowering plants. Imprinted expression is so important in regulating and controlling the embryo development as a typical pattern of epigenetic modification. Gene imprinting primarily occurs in the placenta of mammals and the endosperm of flowering plants, influencing the endosperm development and the seed size finally. Based on upon, we could conjecture that genomic imprinting is related with seeds abortion between inter-ploidy crosses in plant, which is caused by the abnormal endosperm development. Because of M:P=2:1 is the critical ratio of endosperm normal development, imprinting in endosperm is likely to be relevant to its ploidy. So it is a feasible way to verify and research the relation between imprinting and ploidy through creating different ploidy embyros by inter-ploidy crosses in plants. In addition, according to different developmental status between inter-ploidy reciprocal crosses in rice, it has been further analyzed that the expression of genes that acting on imprinting, aleurone cell development, composition of cell wall etc, to determinate if genes expression are affected by changes of ploidy and parent genome proportion or not. Our study has confirmed the "parental conflict theory" in rice and provided a foundation for revealing the relation between imprinting and ploidy.In contrast to the research of imprinting in mammals, study on plant imprinting is fewer. To date, only 22 imprinted genes were identified in A.thaliana, maize and rice endosperm respectively. As a main model plant, rice is very important either in the production practice or in basic researches. This experiment was based on the previous researches which cooperated with Dr. Ming Luo at CSIRO and confirmed more than 200 candidate imprinted genes in rice. We have done inter-ploidy crosses in rice (Nip(2n), H(2n), DH(4n)) and used methods that embyro rescue, ploidy identification, SSR marker, agronomic traits analysis, imprinting analysis and Q-PCR. These are aim to study the imprinting of triploid hybrid seedlings and the expression of endosperm development genes in different ploidy, finally analysis the relation between above results and ploidy The main results are summarized as bellow: 1. Regardless of the background of parent inheritance, there were no normal seeds produced both through the reciprocal crosses between 2n and 4n rice. The early stage of reciprocal seed development was similar to normal hybrid seeds, but the seed was aborted on Dap (day after pollination) 5d and began to shrunken on Dap10d, then was dead finally.2. We have successful obtained all combinations offsprings of Dap 8d, l1d,14d after pollination through embryo rescue and the progeny of Nip(2n)×DH(4n) Dap 20d. The result indicated that hybrid embryo between 2n and 4n rice have the ability to grow up. So we could speculate that the endosperm abnormal development was the reason of seed abort.3. We have identified the hybrid and ploidy of rescued offsprings through SSR and root tip chromosome tabletting. More than 100 triploid offsprings were obtained. There have not shown striking difference between the reciprocal offsprings based on agronomic trait investigation4. The confirmation of expression of imprinted genes in 3n offsprings showed that no imprinting expression in 3n seedlings.5. Q-PCR results have showed that the expression of most genes was striking difference in diverse ploidy endosperm. The gene expression trend was similar in different background reciprocal crosses. These results have indicated that the changes of endosperm ploidy have effected the variation of related genes expression as a main factor.In conclusion, our researches have demonstrated that unequal genome contribution from parents was not the direct reason for imprinting happened. Successful obtaining the triploid offspring proved indirectly that abnormal development of endosperm have caused the seed abortion. On the other hand, expression results of endosperm develop genes have verified the observation of endosperm development in another plant interploidy crosses:when high ploidy was mother, endosperm divided slowly, cellularization occurred early than normal; while as father, endosperm divided quickly and correlated with a later onset of cell-wall fomation.