| Rice, as one of most important crops in the world, serves as the main food for more than half of the world's population, and this number in China even exceeds 60%. Therefore,to substantially increase the unit yield of rice is the only approach to tackle with the contradiction of increasing population and severe decreasing of cultivated land in China.The successful cultivation of hybrid rice in the mid-70s, which is credited as the second green revolution since dwarf gene in rice breeding, has been a great breakthrough in history of word agriculture development and created both huge economic and social benefits.However, statistics indicated that in recent years unit rice yield has reached the bottleneck.On one hand, limited of parental genetic resources to produce hybrid has led to smaller genetic difference between parents, and on the other hand, prevailing reproductive isolation between species and sub-species have severely restricted the utilization of heterosis.Consequently, understand male sterility and reproductive isolation mechanism has great theoretical value and practical meaning to further utilize heterosis.Heterosis utilization started with the discovery of one typical pollen abortion in wild rice. Typical abortion type pollen is produced by the reason that pollen cannot carry on normal meiosis, so understand rice meiosis mechanism can help to better guide and make use of heterosis. Study indicated that African cultivated rice has accumulated many good features by long term artificial selection, such as insect, disease, adversity resistant, and vigorous growth. Therefore, in order to enrich Asian cultivated rice genetic resources,improve yield and break breeding bottleneck, African cultivated rice is the first choice for distant hybridization with Asian cultivated rice. However, there exists severe reproductive isolation in African cultivated rice and Asian cultivated rice hybrid F1, so it is the prerequisite to first deeply comprehend and overcome hybrid sterility before effectively use heterosis between cultivated rice.This paper studies rice male sterility in two aspects. First, rice OsMSH4 gene was cloned through one rice sterile mutant and its function was detailed. Then, interspecies hybrid pollen sterile gene S39 was fine mapped and its pollen abortion process was observed in details. The main content of this study is as below:1. We found a trisomic plant 6537 from anther culture of autotetraploid rice.Compared with normal plant, 6537 had darker, narrower and curling leaves. One extra chromosome was detected by chromosome tabletting, proved that 6537 was typical trisomic plant. From the self-crossed progeny, about 3/4 complete sterile plants were isolated,tentatively named Osmsh4. Osmsh4 mutant pollens cannot be dyed by I2-KI staining, and scanning and transmission electron microscope observation found them to be either smaller or bigger than normal pollens, with irregular shapes and pollen extine ateliosis, but without starch grains, these results demonstrates Osmsh4 mutant pollens are completely abortive.Observation on Osmsh4 mutant embryo sac development through laser scanning confocal microscope found that the mutant could't go through normal meiotic divisions to form the tetrad, thus had neither function megaspore differentiation, nor normal embryo with eight nuclear formation, eventually led to Osmsh4 mutant female gamete also abortion.2. Through observing pollen mother cell chromosome meiosis, it is found that before and during pachytene, there was no obvious difference between chromosome behaviors of Osmsh4 mutant and wild type. While during diplotene, in Osmsh4 mutant pollen mother cell, univalents started to appear and increased significantly in amount when came to diakinesis. From anaphase I to telophase II, random separation of univalents led to unequal chromosomes that move to two poles. Such inequality would inevitably cause genome disorder in the later formed micro spore, thus giving rise to triad or micronucleus.Compared to the 12 bivalents of wild type, Osmsh4 mutant had only 3.95 in average. As chiasmata guaranteed stable bivalent, therefore the reduction of bivalents in mutant might largely cause by fewer chiasmata. Statistics found that wild type averagely has 20.58 chiasmata (n = 81), while Osmsh4 mutant has only 4.51 (n = 152). What's more, in mutant the remaining chiasmata distributed randomly and fit with Poisson distribution. While in wild type, chiasmata distribution severely deviated from Poisson distribution, which indicates that chiasmata interference existed in wild type, but didn't exist in the residual chiasmata of Osmsh4 mutant.3. We cloned OsMSH4 gene by using secondary separation population. Based on sequence alignment, we found OsMSH4 gene to be conservative in other species. Protein analysis showed it has one MUTSd and MUTSac domain, while in Osmsh4; single amino acid substitution exactly took place in the conservative MUTSd domain. Through transgenic complementation and research on OsMSH4 TOS17 mutant, we verified that OsMSH4 was the target gene. qRT-PCR?GUS histochemical staining and in situ hybridization analysis revealed that OsMSH4 was mainly expressed in reproductive organ and the expression reached peak in pollen meiocyte during meiosis, then decreased until finally disappeared. Subcellular localization showed that OsMSH4 protein was principally located in nucleus, which was consistent with its function.4. Previous studies indicated that MSH4 protein can interact with MSH5 protein to form heterodimer, which then combined to Holliday junction for the purpose of stabilize single strand invasion. Through yeast two hybrid (Y2H) and in vitro pull-down, we proved that OsMSH4 could also interact with OsMSH5, and the mutated Osmsh4 lost the ability to interact with OsMSH5. Besides, we also found OsMSH5 could interact with four subunits of OsRPA protein complex, namely OsRPAla, OsRPA2b, OsRPAlc and OsRPA2c. Since OsRPA complex would combine to D-loop and single strand DNA (ssDNA), it meant during meiosis, OsMSH4/OsMSH5 hetrodimer and OsRPA complex interacted with each other to jointly control the second-end capture (SEC) of Holliday junction.5. We develop near-isogenic line (NIL) using Asia cultivated rice dianjingyou (DJY)as receptor and Africa cultivated rice IRGC102295 as donor, and authenticated interspecies pollen sterile gene S39. Cytological observation found that hybrid F1 pollen to be half sterile, belonged to stainable abortive. Abortive pollen grains appeared to be smaller than normal and irregular in shape, accumulated less starch, and had immature exine. Further research detected that the abortive pollen grains came from DJY type in F1,pollen abortion was occurred in the process when microspore transformed from middle stage to later stage.Because of the delay of this process, the DJY type gametes cannot form normal mature pollen and neither can accumulate enough starch.6. Based on map-based cloning, we located S39 gene to a 79-kb region on chromosome 12, where 13 genes were predicted. By sequencing we found two parental genes differentiate obviously in the coding area in this region, what's more, NIL has 82-kb longer sequences than DJY. Further analysis found S39 location section was located in a-400-kb interspecies mutual inversion structure. According to qRT-PCR, several genes in the mapping region showed high expression in anther, however, single gene's transgene experiments didn't get complementary phenotype. Genetic analysis discovered that S39 locus was a commonly existent sterile locus in species and interspecies, and the species of Dular and Kasalath were wide compatible species in this locus. |
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