Analysis Of Genes Regulating Seed Abortion In Inter-ploid Crosses Of Rice

Compared with parents, the offsprings from plant inter-ploid crosses have better phenotypes in drought resistance, disease resistance, insect resistance, plant height, flowering time, growth period and organ size. And plants evolved by polyploidization after distant hybridization. However, inter-ploid rice crosses often fail to produce seeds of normal development, the seeds are shrunken and abortive. The MADS29 gene regulates the plumpness of rice seeds and affects seed development by regulating the degradation of maternal tissues and maintaining the plant's hormone balance. Knock-down of MADS29 results in shriveled and/or aborted seeds. The relationship between MADS29 and seed abortion in inter-ploid crosses of rice was studied by qRT-PCR. It can provide the proofs for the reasonable use of closely related species of rice.Two diploid (2n) and two tetraploid (4n) rice were used in crosses to produce four self-crossed combinations as the control and eight hybridized combinations as reciprocal combinations. Then, pollen grain fertility, pollen germination and seed development were observed. The expressions of MADS29 and related genes were quantified by using qRT-PCR to determine whether these genes were involved in regulation of seed development. The main results are as follows.1. There were no normal seeds produced by 2n and 4n crosses in rice. Observation of the representative seeds of the crosses between 2n and 4n in rice showed that during the third days after pollination (3 DAP), there is no obvious difference in phenotype between those of 2n and 4n. After 6 DAP, the seeds of the reciprocal crosses between 2n and 4n become shrunken, and this phenotype persists until 25 DAP. At later stages, the hybrid seeds with the more severe phenotype show only a layer of skin, and no any inclusions.2. The pollen fertility, germination and elongation of pollen tube were observed showed that:(1) the pollen of rice were vitality in 2n and 4n, and the pollen fertility of 2n was 83.64%, while the 4n was 71.36%. (2) all the pollen of the hybridized and self-crossed combinations after pollination could germinate and went deep into the micropyle of the ovules. These result showed that inter-ploidy cross could be finished double fertilization.3. The observation of the endosperm development after crosses between 2n and 4n in rice by paraffin sections revealed that 3 days after pollination (3 DAP) in 4n×2n cross, the endosperm had entered the cellularization stage, but the number of cells was lower and gathered closely together when compared to the control. However, in the endosperm of seeds of 2n×4n crosses, the cell walls did not form around the nucleus. In addition, some vacuolation was noted in the endosperm, and cellularization was delayed.4. The observation of the morphology of the endosperm starch granules after crosses between 2n and 4n in rice by scanning electron microscopy showed that the seeds produced by 2n selfing showed closely arrange and normal-shaped starch granules. The seeds produced by 4n selfing showed rounder starch granules of intermediate size. While both the seeds produced by 2n×4n and 4n×2n showed smaller and rounder starch granules when compared with those of 2n and 4n, and seeds show abnormal-shaped starch granules.5. Analysis of the expression level of MADS29 in different tissues through RT-PCR and qRT-PCR showed that MADS29 was highly expressed in the developing seed, the gene expression level was 3 DAP>6 DAP>8 DAP), but was not detectable in the vegetative tissues, such as the roots, shoots and leaves, which was consistent with the previous studies, In addition, the relative expression of MADS29 was no difference between 2n and 4n.6. The expression level analysis of MADS29 and related genes in the seeds of different developmental stages from inter-ploid rice crosses between 2n and 4n was carried out by qRT-PCR.The results showed that:(1) the relative expression of MADS29 in hybrid seeds at 6 DAP was significantly higher than those of the control, it was still far higher than those of the control at 8 DAP. And the relative expression of MADS29 in hybrid seeds was 8 DAP>6 DAP. (2) the relative expression of starch synthesis genes (AGPL2?AGPS2b) in hybrid seeds at 6 DAP and 8 DAP was significantly lower than those of the control. The relative expression of starch synthesis genes in those of the control was 8 DAP>6 DAP, while the relative expression of starch synthesis genes in hybrid seeds was 6 DAP>8 DAP. (3) IAA genes (IAA1?IAA19) and programmed cell death (PCD) genes associated with maternal tissues (LOC_Os06g17970, LOC_Os05g31570) in hybrid seeds at 6 DAP and 8 DAP were significantly higher than those of the control.7. The relative expression of MADS29 and starch synthesis genes in hybrid seeds from RNAi-MADS29 Zhonghua 11×DH (4n) and Zhonghua11×DH (4n) were studied by qRT-PCR. The results showed that the relative expression of MADS29 in the seeds produced by RNAi-MADS29 Zhonghua 11×DH was lower than that of zhonghua 11×DH at 6 DAP and 8 DAP, while the relative expression of starch synthesis genes in the seeds produced by RNAi-MADS29 Zhonghua 11×DH was higher than the seeds produced by zhonghua 11×DH at 6 DAP.Above all, the endosperm was abnormally developed in inter-ploid rice crosses between 2n and 4n. And MADS29 was more highly expressed in the seeds of inter-ploid crosses. In addition, the expression of IAA and other genes involved in the PCD of maternal tissues showed up-regulation in the same seeds. However, starch synthesis genes showed down-regulation expression levels. These studies suggest that the gene expression of MADS29 is associated with hybrid seed abortion, it perhaps implys that we can regulate the expression of MADS29 to obtain normal seeds produced by 2n and 4n crosses in rice. Besides, it can provide the proofs for the reasonable use of closely related species of rice.